Lipids and Lipid Compositions for the Delivery of Active Agents

ABSTRACT

This invention provides for a compound of formula (I): 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable salt thereof, wherein R 1 -R 3 , n, p, L 1  and L 2  are defined herein. The compounds of formula (I) and pharmaceutically acceptable salts thereof are cationic lipids useful in the delivery of biologically active agents to cells and tissues.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/104,723, filed Jun. 15, 2016, which is the U.S. National Phase under35 U.S.C. § 371 of International Patent Application No.PCT/US2014/070891, filed Dec. 17, 2014, which claims priority to and thebenefit of U.S. Provisional Application Ser. No. 61/918,941, filed Dec.20, 2013, and U.S. Provisional Application Ser. No. 61/918,182, filedDec. 19, 2013. The entire disclosures of these applications are herebyincorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 14, 2016, isnamed PAT055894-US-PCT_ST25.txt and is 27 KB in size.

FIELD OF THE INVENTION

This invention relates to cationic lipid compounds and to compositionscomprising such compounds. This invention also relates to processes formaking such compounds and compositions, and to methods and uses of suchcompounds and compositions, e.g., to deliver biologically active agents,such as RNA agents, to cells and tissues.

BACKGROUND OF THE INVENTION

The delivery of biologically active agents (including therapeuticallyrelevant compounds) to subjects is often hindered by difficulties in thecompounds reaching the target cell or tissue. In particular, thetrafficking of many biologically active agents into living cells ishighly restricted by the complex membrane systems of the cells. Theserestrictions can result in the need to use much higher concentrations ofbiologically active agents than is desirable to achieve a result, whichincreases the risk of toxic effects and side effects. One solution tothis problem is to utilize specific carrier molecules and carriercompositions which are allowed selective entry into the cell. Lipidcarriers, biodegradable polymers and various conjugate systems can beused to improve delivery of biologically active agents to cells.

One class of biologically active agents that is particularly difficultto deliver to cells is a bio therapeutic (including nucleosides,nucleotides, polynucleotides, nucleic acids and derivatives, such asmRNA, RNAi, and self-replicating RNA agents). In general, nucleic acidsare stable for only a limited duration in cells or plasma. Thedevelopment of RNA interference, RNAi therapy, mRNA therapy, RNA drugs,antisense therapy, gene therapy, and nucleic acid vaccines (e.g., RNAvaccines), among others, has increased the need for an effective meansof introducing active nucleic acid agents into cells. For these reasons,compositions that can stabilize and deliver nucleic acid-based agentsinto cells are of particular interest.

The most well-studied approaches for improving the transport of foreignnucleic acids into cells involve the use of viral vectors orformulations with cationic lipids. Viral vectors can be used to transfergenes efficiently into some cell types, but they generally cannot beused to introduce chemically synthesized molecules into cells.

An alternative approach is to use delivery compositions incorporatingcationic lipids which interact with a biologically active agent at onepart and interact with a membrane system at another part. Suchcompositions are reported to provide liposomes, miscelles, lipoplexes,or lipid nanoparticles, depending on the composition and method ofpreparation (for reviews, see Felgner, 1990, Advanced Drug DeliveryReviews, 5, 162-187; Felgner, 1993, J. Liposome Res., 3, 3-16; Gallas,2013, Chem. Soc. Rev., 42, 7983-7997; Falsini, 2013, J. Med. Chem.dx.doi.org/10.1021/jm400791q; and references therein).

Since the first description of liposomes in 1965 by Bangham (J. Mol.Biol. 13, 238-252), there has been a sustained interest and effort indeveloping lipid-based carrier systems for the delivery of biologicallyactive agents (Allen, 2013, Advanced Drug Delivery Reviews, 65, 36-48).The process of introducing functional nucleic acids into cultured cellsby using positively charged liposomes was first described by PhilipFelgner et al. Proc. Natl. Acad. Sci., USA, 84, 7413-7417 (1987). Theprocess was later demonstrated in vivo by K. L. Brigham et al., Am. J.Med. Sci., 298, 278-281 (1989). More recently, lipid nanoparticleformulations have been developed with demonstrated efficacy in vitro andin vivo. (Falsini, 2013, J. Med. Chem. dx.doi.org/10.1021/jm400791q;Morrissey, 2005, Nat. Biotech., 23, 1002-1007; Zimmerman, 2006, Nature,441, 111-114.; Jayaraman, 2012, Angew. Chem. Int. Ed., 51, 8529-8533.)Lipid formulations are attractive carriers since they can protectbiological molecules from degradation while improving their cellularuptake. Out of the various classes of lipid formulations, formulationswhich contain cationic lipids are commonly used for deliveringpolyanions (e.g. nucleic acids). Such formulations can be formed usingcationic lipids alone and optionally including other lipids andamphiphiles such as phosphatidylethanolamine. It is well known in theart that both the composition of the lipid formulation as well as itsmethod of preparation affect the structure and size of the resultantaggregate (Leung, 2012, J. Phys Chem. C, 116, 18440-18450).

The encapsulation of anionic compounds using cationic lipids isessentially quantitative due to electrostatic interaction. In addition,it is believed that the cationic lipids interact with the negativelycharged cell membranes initiating cellular membrane transport (Akhtar etal., 1992, Trends Cell Bio., 2, 139; Xu et al., 1996, Biochemistry 35,5616). Further, it is believed that the molecular shape, conformation,and properties of the cationic lipids provide enhanced deliveryefficiency from endosomal compartments to the cytosol (Semple, 2010,Nat. Biotech, 28, 172-176; Zhang, 2011, 27, 9473-9483) Although the useof cationic lipids for cellular delivery of biologically active agentshas been shown to have several advantages, there still remains a needfor further cationic lipids that facilitate the systemic and localdelivery of biologically active agents such as mRNA and RNAi agents tocells. There is also a need for cationic lipids that, relative to thosecationic lipids that are known in the art, improve the systemic andlocal delivery of biologically active agents to cells. There is afurther need for lipid formulations that have optimized physicalcharacteristics for improved systemic and local delivery of biologicallyactive agents to specific organs and to tumors, especially tumorsoutside the liver.

In addition, there is a need for further cationic lipids that providedecreased toxicity (or improved therapeutic index), relative to thosecationic lipids that are known in the art.

Traditional cationic lipids have been employed for RNA and DNA deliveryto the liver or tumors but suffer from non-optimal delivery efficiencyalong with tissue and organ toxicity at higher doses. One method ofreducing exposure and increasing biocompatability of cationic lipids isto incorporate chemically or biochemically degradable functionalities,(such as ester, amide, acetal, imine, etc.), which can lead to enhancedin vivo clearance (Maier, 2013, 21, 1570-1578).

SUMMARY OF THE INVENTION

The present invention provides a cationic lipid scaffold thatdemonstrates enhanced efficacy along with lower toxicity (improvedtherapeutic index) as a result of lower sustained lipid levels in therelavent tissues, and for local delivery applications (eye, ear, skin,lung); delivery to muscle (i.m.), fat, or sub cutaneous cells (s.c.dosing).

In one aspect, this invention provides for a compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein R¹-R³, n, p, L₁and L₂ are defined herein. The compounds of formula (I) andpharmaceutically acceptable salts thereof are cationic lipids useful inthe delivery of biologically active agents to cells and tissues.

In a second aspect, this invention provides for a lipid compositioncomprising a compound according to formula (I) (i.e. a lipid compositionof the invention), or a pharmaceutically acceptable salt thereof. In oneembodiment, the lipid composition further comprises at least one otherlipid component. In another embodiment, the lipid composition furthercomprises a biologically active agent, optionally in combination with onone more other lipid components. In another embodiment the lipidcomposition is in the form of a liposome. In another embodiment thelipid composition is in the form of a lipid nanoparticle (LNP). Inanother embodiment the lipid composition is suitable for delivery to theliver. In another embodiment the lipid composition is suitable fordelivery to a tumor. In another embodiment the lipid composition issuitable for immunization purposes. In another embodiment the lipidcomposition is suitable for local delivery applications (eye, ear, skin,lung); delivery to muscle (i.m.), fat, or sub cutaneous cells (s.c.dosing).

In a third aspect, this invention provides for a pharmaceuticalcomposition (i.e. formulation) comprising a lipid composition of theinvention and a pharmaceutically acceptable carrier or excipient. In oneembodiment, the pharmaceutical composition comprises at least one otherlipid component in the lipid composition. In another embodiment thelipid composition is in the form of a liposome. In another embodimentthe lipid composition is in the form of a lipid nanoparticle. In anotherembodiment the lipid composition is suitable for delivery to the liver.In another embodiment the lipid composition is suitable for delivery toa tumor. In another embodiment the lipid composition is suitable forlocal delivery applications (eye, ear, skin, lung); delivery to muscle(i.m.), fat, or sub cutaneous cells (s.c. dosing). In another embodimentthe biologically active agent is an RNA or DNA. In another embodimentthe lipid composition is suitable for immunization purposes, and thebiologically active agent is a RNA or DNA which encodes an immunogen.

In a fourth aspect, this invention provides a method for the treatmentof a disease or condition comprising the step of administering atherapeutically effective amount of a lipid composition of the inventionto a patient in need of treatment thereof. In one embodiment, thedisease or condition is treatable by administering an RNA or DNA agent.In another embodiment the lipid composition is suitable for immunizationpurposes, and the biologically active agent is an RNA or DNA whichencodes an immunogen.

In a fifth aspect, this invention provides for the use of a lipidcomposition of the invention in treating a disease or condition in apatient. In one embodiment, the disease or condition is treatable byadministering an RNA or DNA agent.

In a sixth aspect, this invention provides a method for inducing animmune response in a subject against an immunogen of interest comprisingadministering an immunologically effective amount of a lipid compositionof the invention to the subject, in combination with a RNA or DNA thatencodes the immunogen.

In a seventh aspect, this invention provides for the use of a lipidcomposition of the invention in inducing an immune response in a subjectagainst an immunogen of interest (e.g., in the preparation ormanufacture of a medicament). The lipid is used in combination with aRNA which encodes an immunogen.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments will be further described in connection with theattached figure. It is intended that the drawing included as part of thespecification be illustrative of the embodiments and should in no way beconsidered as a limitation on the scope of the invention.

FIG. 1 shows a synthetic scheme, Scheme 1, which illustrates apreparation of final compounds of formula (I).

DETAILED DESCRIPTION OF THE INVENTION

In a first embodiment, the invention is a compound, or salt thereof, offormula (I) (a “lipid provided by the invention”):

Wherein n is 0, 1, 2, 3 or 4; p is 0, 1, 2, 3, 4, 5, 6, 7 or 8; L₁ is—O— or a bond; L₂ is —OC(O)— or —C(O)O—; R¹ is selected from:

v is 0, 1, 2, 3 or 4; w is 0, 1, 2, or 3; Cyl is 5-7 membered nitrogencontaining heterocycle optionally substituted with one or two alkylgroups; Ar is an aryl group, optionally substituted with C₁₋₈ alkylamino group; R and R′ are each, independently, hydrogen or C₁₋₈ alkyl;andR² is selected from C₆₋₂₀ alkyl optionally substituted with a hydroxyl,C₁₅₋₁₉ alkenyl, C₁₋₁₂alkyl-OC(O)—C₅₋₂₀alkyl, C₁₋₁₂alkyl-C(O)O—C₅₋₂₀alkyland

R³ is selected from: C₄₋₂₂ alkyl, C₁₂₋₂₂ alkenyl,

In a second embodiment, the invention is a compound, or salt thereof,according to the first embodiment, wherein the compound is of formula(I):

n is 0, 1, 2, 3 or 4; p is 0, 1, 2, 3, 4, 5, 6, 7 or 8; L₁ is —O— or abond; L₂ is —OC(O)— or —C(O)O—; R¹ is selected from:

v is 0, 1, 2, 3 or 4; w is 0, 1, 2, or 3; Cyl is 5-7 membered nitrogencontaining heterocycle optionally substituted with one or two alkylgroups; R and R′ are each, independently, hydrogen or C₁₋₈ alkyl; and R²is selected from C₆₋₂₀ alkyl optionally substituted with a hydroxyl,C₁₅₋₁₉ alkenyl, C₁₋₁₂alkyl-OC(O)—C₅₋₂₀alkyl, C₁₋₁₂alkyl-C(O)O—C₅₋₂₀alkyland

R³ is selected from: C₄₋₂₂ alkyl, C₁₂₋₂₂ alkenyl,

In a third embodiment, the invention is the compound, or salt thereof,according to the first or second embodiments, wherein the compound is offormula (II):

In a fourth embodiment, the invention is the compound, or salt thereof,according to any one of the first through third embodiments, wherein thecompound is of formula (III):

In a fifth embodiment, the invention is the compound, or salt thereof,according to any one of the first through fourth embodiments, wherein R²is selected from:

In a sixth embodiment, the invention is the compound, or salt thereof,according to any one of the first through fifth embodiments, wherein R²is selected from:

In a seventh embodiment, the invention is the compound, or salt thereof,according to any one of the first through sixth embodiments, wherein R²is

In an eighth embodiment, the invention is the compound, or salt thereof,of any one of the first through seventh embodiments, wherein thecompound is of formula (IV):

In a ninth embodiment, the invention is the compound, or salt thereof,according to any one of the first through eighth embodiments, wherein R³is selected from:

In a tenth embodiment, the invention is the compound, or salt thereof,according to any one of the first through ninth embodiments, wherein R³is selected from:

In an eleventh embodiment, the invention is the compound, or saltthereof, according to any one of the first through tenth embodiments,wherein R³ is

In a twelfth embodiment, the invention is the compound, or salt thereof,according to any one of the first through eleventh embodiments, whereinthe compound is of formula (V):

In a thirteenth embodiment, the invention is the compound, or saltthereof, according to any one of the first through twelfth embodiments,wherein the compound is of formula (VI):

In a fourteenth embodiment, the invention is the compound, or saltthereof, according to any one of the first through thirteenthembodiments, wherein the compound is of formula (VII):

In a fifteenth embodiment, the invention is the compound, or saltthereof, of any one of the first through fourteenth embodiments, whereinR¹ is selected from:

In a sixteenth embodiment, the invention is the compound, or saltthereof, of any one of of the first through fifteenth embodiments,wherein R¹ is selected from:

In a seventeenth embodiment, the invention is the compound, or saltthereof, of any one of the first through sixteenth embodiments, whereinR¹ is

In an eighteenth embodiment, the invention is the compound, or saltthereof, according to any one of the first through seventeenthembodiments, wherein the compound is selected from:

-   2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azaoctadecan-18-yl)propane-1,3-diyl    dioctanoate;-   2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2-azaheptadecan-17-yl)propane-1,3-diyl    dioctanoate;-   2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2-azapentadecan-15-yl)propane-1,3-diyl    dioctanoate;-   2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azahexadecan-16-yl)propane-1,3-diyl    dioctanoate;-   2-(8-dodecyl-2-methyl-6,12-dioxo-5,7,11-trioxa-2-azaheptadecan-17-yl)propane-1,3-diyl    dioctanoate;-   2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azanonadecan-19-yl)propane-1,3-diyl    dioctanoate;-   2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2-azaoctadecan-18-yl)propane-1,3-diyl    dioctanoate;-   2-(8-dodecyl-2-methyl-6,12-dioxo-5,7,11-trioxa-2-azaoctadecan-18-yl)propane-1,3-diyl    dioctanoate;-   2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azaicosan-20-yl)propane-1,3-diyl    dioctanoate;-   2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2-azanonadecan-19-yl)propane-1,3-diyl    dioctanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    4,4-bis(octyloxy)butanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    4,4-bis((2-ethylhexyl)oxy)butanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    4,4-bis((2-propylpentyl)oxy)butanoate;-   3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl    4,4-bis((2-propylpentyl)oxy)butanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    4,4-bis((2-propylpentyl)oxy)butanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    6,6-bis(octyloxy)hexanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    6,6-bis(hexyloxy)hexanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    6,6-bis((2-ethylhexyl)oxy)hexanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    8,8-bis(hexyloxy)octanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    8,8-dibutoxyoctanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    8,8-bis((2-propylpentyl)oxy)octanoate;-   3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl    8,8-bis((2-propylpentyl)oxy)octanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    8,8-bis((2-propylpentyl)oxy)octanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    3-octylundecanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    3-octylundec-2-enoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    7-hexyltridec-6-enoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    9-pentyltetradecanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    9-pentyltetradec-8-enoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    5-heptyldodecanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)tridecyl    5-heptyldodecanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)undecyl    5-heptyldodecanoate;-   1,3-bis(octanoyloxy)propan-2-yl    (3-(((2-(dimethylamino)ethoxy)carbonyl)oxy)pentadecyl) succinate;-   1,3-bis(octanoyloxy)propan-2-yl    (3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl) succinate;-   1-(3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl) 10-octyl    decanedioate;-   1-(3-(((3-(diethylamino) propoxy)carbonyl)oxy)pentadecyl) 10-octyl    decanedioate;-   1-(3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl)    10-octyl decanedioate;-   1-(3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl)    10-(2-ethylhexyl) decanedioate;-   1-(3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl)    10-(2-ethylhexyl) decanedioate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    10-(octanoyloxy)decanoate;-   8-dodecyl-2-methyl-6,12-dioxo-5,7,11-trioxa-2-azanonadecan-19-yl    decanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    10-(octanoyloxy)decanoate;-   3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl    10-(octanoyloxy)decanoate;-   (9Z,12Z)-3-(((3-(di methylamino)propoxy)carbonyl)oxy)pentadecyl    octadeca-9,12-dienoate;-   (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    octadeca-9,12-dienoate;-   (9Z,12Z)-3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl    octadeca-9,12-dienoate;-   (9Z,12Z)-3-(((2-(dimethylamino)ethoxy)carbonyl)oxy)pentadecyl    octadeca-9,12-dienoate;-   1-((9Z,12Z)-octadeca-9,12-dienoyloxy) pentadecan-3-yl    1,4-dimethylpiperidine-4-carboxylate;-   2-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecyl    4,4-bis((2-ethylhexyl)oxy)butanoate;-   (9Z,12Z)-(12Z,15Z)-3-((3-(dimethylamino)propanoyl)oxy)henicosa-12,15-dien-1-yl    octadeca-9,12-dienoate;-   (12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)henicosa-12,15-dien-1-yl    3-octylundecanoate;-   (12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)henicosa-12,15-dien-1-yl    5-heptyldodecanoate;-   (12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)henicosa-12,15-dien-1-yl    7-hexyltridecanoate;-   (12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)henicosa-12,15-dien-1-yl    9-pentyltetradecanoate;-   (12Z,15Z)-1-((((9Z,12Z)-octadeca-9,12-dien-1-yloxy)carbonyl)oxy)    henicosa-12,15-dien-3-yl 3-(dimethylamino)propanoate;-   (13Z,16Z)-4-(((2-(dimethylamino)ethoxy)carbonyl)oxy)docosa-13,16-dien-1-yl    2,2-bis(heptyloxy)acetate;-   (13Z,16Z)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)docosa-13,16-dien-1-yl    2,2-bis(heptyloxy)acetate;-   2,2-bis(heptyloxy)ethyl    3-((3-ethyl-10-((9Z,12Z)-octadeca-9,12-dien-1-yl)-8,15-dioxo-7,9,14-trioxa-3-azaheptadecan-17-yl)disulfanyl)propanoate;-   (13Z,16Z)-4-(((3-(dimethylamino)propoxy)carbonyl)oxy)docosa-13,16-dien-1-yl    heptadecan-9-yl succinate;-   (9Z,12Z)-2-(((11Z,14Z)-2-((3-(dimethylamino)propanoyl)oxy)icosa-11,14-dien-1-yl)oxy)ethyl    octadeca-9,12-dienoate;-   (9Z,12Z)-3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl    octadeca-9,12-dienoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl    3-octylundecanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-hydroxytridecyl    5-heptyldodecanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl    5-heptyldodecanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl    7-hexyltridecanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-hydroxytridecyl    9-pentyltetradecanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl    9-pentyltetradecanoate;-   1-(3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl)    10-octyl decanedioate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl    10-(octanoyloxy)decanoate;-   (9Z,12Z)-3-(((3-(di    methylamino)propoxy)carbonyl)oxy)-5-octyltridecyl    octadeca-9,12-dienoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-5-octyltridecyl    decanoate;-   5-(((3-(dimethylamino)propoxy)carbonyl)oxy)-7-octylpentadecyl    octanoate;-   (9Z,12Z)-5-(((3-(dimethylamino)    propoxy)carbonyl)oxy)-7-octylpentadecyl octadeca-9,12-dienoate;-   9-(((3-(dimethylamino)propoxy)carbonyl)oxy)-11-octylnonadecyl    octanoate;-   9-(((3-(dimethylamino)propoxy)carbonyl)oxy)-11-octylnonadecyl    decanoate;-   (9Z,12Z)-9-(((3-(di methylamino)propoxy)carbonyl)oxy)nonadecyl    octadeca-9,12-dienoate;-   9-(((3-(dimethylamino)propoxy)carbonyl)oxy)nonadecyl hexanoate;-   9-(((3-(dimethylamino)propoxy)carbonyl)oxy)nonadecyl    3-octylundecanoate;-   9-((4-(dimethylamino)butanoyl)oxy) nonadecyl hexanoate;-   9-((4-(dimethylamino)butanoyl)oxy) nonadecyl 3-octylundecanoate;-   (9Z,9′Z,12Z,12′Z)-2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl    bis(octadeca-9,12-dienoate);-   (9Z,9′Z,12Z,12′Z)-2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)    hexadecanoyl)oxy)propane-1,3-diyl bis(octadeca-9,12-dienoate);-   (9Z,9′Z,12Z,12′Z,15Z,15′Z)-2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)    hexadecanoyl)oxy)propane-1,3-diyl bis(octadeca-9,12,15-trienoate);-   (Z)-2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)    hexadecanoyl)oxy)propane-1,3-diyl dioleate;-   2-((4-(((3-(diethylamino) propoxy)carbonyl)oxy)    hexadecanoyl)oxy)propane-1,3-diyl ditetradecanoate;-   2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl    ditetradecanoate;-   2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)    propane-1,3-diyl ditetradecanoate;-   2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl    didodecanoate;-   2-((4-(((3-(diethylamino) propoxy)carbonyl)oxy)    hexadecanoyl)oxy)propane-1,3-diyl didodecanoate;-   2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)    propane-1,3-diyl didodecanoate;-   2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)    hexadecanoyl)oxy)propane-1,3-diyl bis(decanoate);-   2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)    propane-1,3-diyl bis(decanoate);-   2-((4-(((3-(diethylamino) propoxy)carbonyl)oxy)    hexadecanoyl)oxy)propane-1,3-diyl dioctanoate;-   2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)    propane-1,3-diyl dioctanoate;-   2-(((13Z,16Z)-4-(((3-(dimethylamino)propoxy)carbonyl)oxy)docosa-13,16-dienoyl)oxy)propane-1,3-diyl    dioctanoate;-   2-(((13Z,16Z)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)docosa-13,16-dienoyl)oxy)propane-1,3-diyl    dioctanoate;-   (9Z,9′Z,12Z,12′Z)-2-((2-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecanoyl)oxy)    propane-1,3-diyl bis(octadeca-9,12-dienoate);-   (9Z,9′Z,12Z,12′Z)-2-((2-(((3-(dimethylamino)propoxy)carbonyl)oxy)dodecanoyl)oxy)propane-1,3-diyl    bis(octadeca-9,12-dienoate);-   (9Z,9′Z,12Z,12′Z)-2-((2-(((3-(dimethylamino)propoxy)carbonyl)oxy)tetradecanoyl)oxy)propane-1,3-diyl    bis(octadeca-9,12-dienoate);-   (9Z,9′Z,12Z,12′Z)-2-((2-(((3-(diethylamino)propoxy)carbonyl)oxy)dodecanoyl)oxy)propane-1,3-diyl    bis(octadeca-9,12-dienoate);-   2-((2-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecanoyl)oxy)propane-1,3-diyl    dioctanoate;-   4,4-bis(octyloxy)butyl 4-(((3-(dimethylamino)propoxy)carbonyl)oxy)    hexadecanoate;-   4,4-bis(octyloxy)butyl 2-(((3-(diethylamino)    propoxy)carbonyl)oxy)dodecanoate;-   (9Z,12Z)-10-dodecyl-3-ethyl-14-(2-((9Z,12Z)-octadeca-9,12-dienoyloxy)ethyl)-8,13-dioxo-7,9-dioxa-3,14-diazahexadecan-16-yl    octadeca-9,12-dienoate;-   2-((4-(((3-(diethylamino)    propoxy)carbonyl)oxy)-11-(octanoyloxy)undecanoyl)oxy)propane-1,3-diyl    dioctanoate;-   (9Z,9′Z,12Z,12′Z)-2-(9-dodecyl-2-methyl-7,12-dioxo-6,8,13-trioxa-2-azatetradecan-14-yl)propane-1,3-diyl    bis(octadeca-9,12-dienoate);-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    4,4-bis(octyloxy)butanoate;-   3-(((3-(piperidin-1-yl)propoxy)carbonyl)oxy)pentadecyl    6,6-bis(octyloxy)hexanoate;-   3-(((3-(piperazin-1-yl)propoxy)carbonyl)oxy)pentadecyl    6,6-bis(octyloxy)hexanoate;-   3-(((4-(diethylamino) butoxy)carbonyl)oxy)pentadecyl    6,6-bis(octyloxy)hexanoate;-   3-(((3-(4-methylpiperazin-1-yl)propoxy)carbonyl)oxy)pentadecyl    6,6-bis(octyloxy)hexanoate;-   3-((((1-methylpiperidin-4-yl)methoxy)carbonyl)oxy)pentadecyl    6,6-bis(octyloxy)hexanoate;-   3-(((3-morpholinopropoxy)carbonyl)oxy) pentadecyl 6,6-bis(octyloxy)    hexanoate;-   3-(((2-(diethylamino)ethoxy)carbonyl)oxy)pentadecyl    6,6-bis(octyloxy)hexanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    6,6-bis(octyloxy)hexanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    6,6-bis((2-propylpentyl)oxy)hexanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    6,6-bis((2-propylpentyl)oxy)hexanoate LXR420:    3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    6,6-bis((3-ethylpentyl)oxy)hexanoate;-   (2R)-1-((6,6-bis(octyloxy)hexanoyl)oxy) pentadecan-3-yl 1-methyl    pyrrolidi ne-2-carboxylate;-   (2S)-1-((6,6-bis(octyloxy)hexanoyl)oxy)pentadecan-3-yl    1-methylpyrrolidine-2-carboxylate;-   (2R)-1-((6,6-bis(octyloxy) hexanoyl)oxy)pentadecan-3-yl    pyrrolidine-2-carboxylate;-   1-((6,6-bis(octyloxy)hexanoyl)oxy) pentadecan-3-yl    1,3-dimethylpyrrolidine-3-carboxylate;-   3-((3-(1-methylpiperidin-4-yl)propanoyl)oxy)pentadecyl    6,6-bis(octyloxy)hexanoate;-   1-((6,6-bis(octyloxy)hexanoyl)oxy) pentadecan-3-yl    1,4-dimethylpiperidine-4-carboxylate;-   3-((5-(diethylamino)pentanoyl)oxy) pentadecyl 6,6-bis(octyloxy)    hexanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    5-(4,6-diheptyl-1,3-dioxan-2-yl)pentanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)undecyl    6,6-bis(octyloxy)hexanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)tridecyl    6,6-bis(octyloxy)hexanoate;-   (12Z,15Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)henicosa-12,15-dien-1-yl    6,6-bis(octyloxy)hexanoate;-   6-((6,6-bis(octyloxy)hexanoyl)oxy)-4-(((3-(diethylamino)    propoxy)carbonyl)oxy)hexyl octanoate;-   4,4-bis(octyloxy)butyl    5-(((3-(diethylamino)propoxy)carbonyl)oxy)heptadecanoate;-   4,4-bis(octyloxy)butyl (3-(diethylamino)propyl) pentadecane-1,3-diyl    dicarbonate;-   2-(5-((4-((1,4-dimethylpiperidine-4-carbonyl)oxy)    hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyl dioctanoate;-   2-(5-((4-((1,3-dimethylpyrrolidine-3-carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyl    dioctanoate;-   2-(5-oxo-5-((4-(((S)-pyrrolidine-2-carbonyl)oxy)hexadecyl)oxy)pentyl)    propane-1,3-diyl dioctanoate;-   2-(5-((4-(((((S)-1-methyl    pyrrolidin-3-yl)oxy)carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyl    dioctanoate;-   2-(5-((4-(((((R)-1-methylpyrrolidin-3-yl)oxy)carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyl    dioctanoate;-   2-(5-((4-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)    hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyl dioctanoate;-   2-(5-((4-((((1-methylpiperidin-4-yl)oxy)carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyl    dioctanoate;-   2-(10-dodecyl-3-ethyl-8,15-dioxo-7,9,14-trioxa-3-azanonadecan-19-yl)propane-1,3-diyl    dioctanoate;-   2-(11-dodecyl-3-ethyl-9,15-dioxo-8,10,14-trioxa-3-azanonadecan-19-yl)propane-1,3-diyl    dioctanoate;-   2-(5-((3-(((3-(1H-imidazol-1-yl)propoxy)carbonyl)oxy)pentadecyl)oxy)-5-oxopentyl)propane-1,3-diyl    dioctanoate;-   2-(5-oxo-5-((3-(((3-(piperidin-1-yl)propoxy)carbonyl)oxy)pentadecyl)oxy)pentyl)propane-1,3-diyl    dioctanoate; and-   2-(12-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azaoctadecan-18-yl)propane-1,3-diyl    dioctanoate.

In a nineteenth embodiment, the invention is the compound, or saltthereof, according to any one of the first through eighteenthembodiments, wherein the compound is selected from:

-   2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azaoctadecan-18-yl)propane-1,3-diyl    dioctanoate;-   2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2-azaheptadecan-17-yl)propane-1,3-diyl    dioctanoate;-   2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2-azapentadecan-15-yl)propane-1,3-diyl    dioctanoate;-   2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azahexadecan-16-yl)propane-1,3-diyl    dioctanoate;-   2-(8-dodecyl-2-methyl-6,12-dioxo-5,7,11-trioxa-2-azaheptadecan-17-yl)propane-1,3-diyl    dioctanoate;-   2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azanonadecan-19-yl)propane-1,3-diyl    dioctanoate;-   2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2-azaoctadecan-18-yl)propane-1,3-diyl    dioctanoate;-   2-(8-dodecyl-2-methyl-6,12-dioxo-5,7,11-trioxa-2-azaoctadecan-18-yl)propane-1,3-diyl    dioctanoate;-   2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azaicosan-20-yl)propane-1,3-diyl    dioctanoate;-   2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2-azanonadecan-19-yl)propane-1,3-diyl    dioctanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    4,4-bis(octyloxy)butanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    4,4-bis((2-ethylhexyl)oxy)butanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    4,4-bis((2-propylpentyl)oxy)butanoate;-   3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl    4,4-bis((2-propylpentyl)oxy)butanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    4,4-bis((2-propylpentyl)oxy)butanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    6,6-bis(octyloxy)hexanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    6,6-bis(hexyloxy)hexanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    6,6-bis((2-ethylhexyl)oxy)hexanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    8,8-bis(hexyloxy)octanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    8,8-dibutoxyoctanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    8,8-bis((2-propylpentyl)oxy)octanoate;-   3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl    8,8-bis((2-propylpentyl)oxy)octanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    8,8-bis((2-propylpentyl)oxy)octanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    3-octylundecanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    3-octylundec-2-enoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    7-hexyltridec-6-enoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    9-pentyltetradecanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    9-pentyltetradec-8-enoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    5-heptyldodecanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)tridecyl    5-heptyldodecanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)undecyl    5-heptyldodecanoate;-   1,3-bis(octanoyloxy)propan-2-yl    (3-(((2-(dimethylamino)ethoxy)carbonyl)oxy)pentadecyl) succinate;-   1,3-bis(octanoyloxy)propan-2-yl    (3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl) succinate;-   1-(3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl) 10-octyl    decanedioate;-   1-(3-(((3-(diethylamino) propoxy)carbonyl)oxy)pentadecyl) 10-octyl    decanedioate;-   1-(3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl)    10-octyl decanedioate;-   1-(3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl)    10-(2-ethylhexyl) decanedioate;-   1-(3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl)    10-(2-ethylhexyl) decanedioate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl    10-(octanoyloxy)decanoate;-   8-dodecyl-2-methyl-6,12-dioxo-5,7,11-trioxa-2-azanonadecan-19-yl    decanoate;-   3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    10-(octanoyloxy)decanoate;-   3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl    10-(octanoyloxy)decanoate;-   (9Z,12Z)-3-(((3-(di methylamino)propoxy)carbonyl)oxy)pentadecyl    octadeca-9,12-dienoate;-   (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl    octadeca-9,12-dienoate;-   (9Z,12Z)-3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl    octadeca-9,12-dienoate;-   (9Z,12Z)-3-(((2-(dimethylamino)ethoxy)carbonyl)oxy)pentadecyl    octadeca-9,12-dienoate;-   1-((9Z,12Z)-octadeca-9,12-dienoyloxy) pentadecan-3-yl    1,4-dimethylpiperidine-4-carboxylate;-   2-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecyl    4,4-bis((2-ethylhexyl)oxy)butanoate;-   (9Z,12Z)-(12Z,15Z)-3-((3-(dimethylamino)propanoyl)oxy)henicosa-12,15-dien-1-yl    octadeca-9,12-dienoate;-   (12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)henicosa-12,15-dien-1-yl    3-octylundecanoate;-   (12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)henicosa-12,15-dien-1-yl    5-heptyldodecanoate;-   (12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)henicosa-12,15-dien-1-yl    7-hexyltridecanoate;-   (12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)henicosa-12,15-dien-1-yl    9-pentyltetradecanoate;-   (12Z,15Z)-1-((((9Z,12Z)-octadeca-9,12-dien-1-yloxy)carbonyl)oxy)    henicosa-12,15-dien-3-yl 3-(dimethylamino)propanoate;-   (13Z,16Z)-4-(((2-(dimethylamino)ethoxy)carbonyl)oxy)docosa-13,16-dien-1-yl    2,2-bis(heptyloxy)acetate;-   (13Z,16Z)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)docosa-13,16-dien-1-yl    2,2-bis(heptyloxy)acetate;-   2,2-bis(heptyloxy)ethyl    3-((3-ethyl-10-((9Z,12Z)-octadeca-9,12-dien-1-yl)-8,15-dioxo-7,9,14-trioxa-3-azaheptadecan-17-yl)disulfanyl)propanoate;-   (13Z,16Z)-4-(((3-(dimethylamino)propoxy)carbonyl)oxy)docosa-13,16-dien-1-yl    heptadecan-9-yl succinate;-   (9Z,12Z)-2-(((11Z,14Z)-2-((3-(dimethylamino)propanoyl)oxy)icosa-11,14-dien-1-yl)oxy)ethyl    octadeca-9,12-dienoate;-   (9Z,12Z)-3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl    octadeca-9,12-dienoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl    3-octylundecanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-hydroxytridecyl    5-heptyldodecanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl    5-heptyldodecanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl    7-hexyltridecanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-hydroxytridecyl    9-pentyltetradecanoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl    9-pentyltetradecanoate;-   1-(3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl)    10-octyl decanedioate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl    10-(octanoyloxy)decanoate;-   (9Z,12Z)-3-(((3-(di    methylamino)propoxy)carbonyl)oxy)-5-octyltridecyl    octadeca-9,12-dienoate;-   3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-5-octyltridecyl    decanoate;-   5-(((3-(dimethylamino)propoxy)carbonyl)oxy)-7-octylpentadecyl    octanoate;-   (9Z,12Z)-5-(((3-(dimethylamino)    propoxy)carbonyl)oxy)-7-octylpentadecyl octadeca-9,12-dienoate;-   9-(((3-(dimethylamino)propoxy)carbonyl)oxy)-11-octylnonadecyl    octanoate;-   9-(((3-(dimethylamino)propoxy)carbonyl)oxy)-11-octylnonadecyl    decanoate;-   (9Z,12Z)-9-(((3-(di methylamino)propoxy)carbonyl)oxy)nonadecyl    octadeca-9,12-dienoate;-   9-(((3-(dimethylamino)propoxy)carbonyl)oxy)nonadecyl hexanoate;-   9-(((3-(dimethylamino)propoxy)carbonyl)oxy)nonadecyl    3-octylundecanoate;-   9-((4-(dimethylamino)butanoyl)oxy) nonadecyl hexanoate;-   9-((4-(dimethylamino)butanoyl)oxy) nonadecyl 3-octylundecanoate;-   (9Z,9′Z,12Z,12′Z)-2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl    bis(octadeca-9,12-dienoate);-   (9Z,9′Z,12Z,12′Z)-2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)    hexadecanoyl)oxy)propane-1,3-diyl bis(octadeca-9,12-dienoate);-   (9Z,9′Z,12Z,12′Z,15Z,15′Z)-2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)    hexadecanoyl)oxy)propane-1,3-diyl bis(octadeca-9,12,15-trienoate);-   (Z)-2-((4-(((3-(dimethylamino) propoxy)carbonyl)oxy)    hexadecanoyl)oxy)propane-1,3-diyl dioleate;-   2-((4-(((3-(diethylamino) propoxy)carbonyl)oxy)    hexadecanoyl)oxy)propane-1,3-diyl ditetradecanoate;-   2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl    ditetradecanoate;-   2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)    propane-1,3-diyl ditetradecanoate;-   2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl    didodecanoate;-   2-((4-(((3-(diethylamino) propoxy)carbonyl)oxy)    hexadecanoyl)oxy)propane-1,3-diyl didodecanoate;-   2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)    propane-1,3-diyl didodecanoate;-   2-((4-(((3-(diethylamino) propoxy)carbonyl)oxy)    hexadecanoyl)oxy)propane-1,3-diyl bis(decanoate);-   2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)    propane-1,3-diyl bis(decanoate);-   2-((4-(((3-(diethylamino) propoxy)carbonyl)oxy)    hexadecanoyl)oxy)propane-1,3-diyl dioctanoate;-   2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)    propane-1,3-diyl dioctanoate;-   2-(((13Z,16Z)-4-(((3-(dimethylamino)propoxy)carbonyl)oxy)docosa-13,16-dienoyl)oxy)propane-1,3-diyl    dioctanoate;-   2-(((13Z,16Z)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)docosa-13,16-dienoyl)oxy)propane-1,3-diyl    dioctanoate;-   (9Z,9′Z,12Z,12′Z)-2-((2-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecanoyl)oxy)    propane-1,3-diyl bis(octadeca-9,12-dienoate);-   (9Z,9′Z,12Z,12′Z)-2-((2-(((3-(dimethylamino)propoxy)carbonyl)oxy)dodecanoyl)oxy)propane-1,3-diyl    bis(octadeca-9,12-dienoate);-   (9Z,9′Z,12Z,12′Z)-2-((2-(((3-(dimethylamino)propoxy)carbonyl)oxy)tetradecanoyl)oxy)propane-1,3-diyl    bis(octadeca-9,12-dienoate);-   (9Z,9′Z,12Z,12′Z)-2-((2-(((3-(diethylamino)propoxy)carbonyl)oxy)dodecanoyl)oxy)propane-1,3-diyl    bis(octadeca-9,12-dienoate);-   2-((2-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecanoyl)oxy)propane-1,3-diyl    dioctanoate;-   4,4-bis(octyloxy)butyl 4-(((3-(dimethylamino)propoxy)carbonyl)oxy)    hexadecanoate;-   4,4-bis(octyloxy)butyl 2-(((3-(diethylamino)    propoxy)carbonyl)oxy)dodecanoate;-   (9Z,12Z)-10-dodecyl-3-ethyl-14-(2-((9Z,12Z)-octadeca-9,12-dienoyloxy)ethyl)-8,13-dioxo-7,9-dioxa-3,14-diazahexadecan-16-yl    octadeca-9,12-dienoate;-   2-((4-(((3-(diethylamino)    propoxy)carbonyl)oxy)-11-(octanoyloxy)undecanoyl)oxy)propane-1,3-diyl    dioctanoate; and-   (9Z,9′Z,12Z,12′Z)-2-(9-dodecyl-2-methyl-7,12-dioxo-6,8,13-trioxa-2-azatetradecan-14-yl)propane-1,3-diyl    bis(octadeca-9,12-dienoate).

In a twentieth embodiment, the invention is a lipid compositioncomprising a compound according to anyone of the first throughnineteenth embodiments, or a pharmaceutically acceptable salt thereof.

In a twentyfirst embodiment, the invention is the lipid compositionaccording to the twentieth embodiment further comprising a biologicallyactive agent.

In a twentysecond embodiment, the invention is the lipid compositionaccording to the twentyfirst embodiment, wherein the biologically activeagent is a nucleic acid.

In a twentythird embodiment, the invention is the lipid compositionaccording to any one of the twentyfirst or twentysecond embodiments,wherein the biologically active agent is a DNA, siRNA or mRNA.

In a twentyfourth embodiment, the invention is the lipid compositionaccording to any one of the twentyfirst through twentythird embodiments,wherein the biologically active agent is a mRNA.

In a twentyfifth embodiment, the invention is the lipid compositionaccording to any one of the twentyfirst through twentythird embodiments,wherein the biologically active agent is a siRNA.

In a twentysixth embodiment, the invention is the lipid compositionaccording to any one of the twentieth through twentyfifth embodiments,further comprising a helper lipid.

In a twentyseventh embodiment, the invention is the lipid compositionaccording to any one of the twentieth through twentysixth embodimentsfurther comprising a neutral lipid.

In a twentyeighth embodiment, the invention is the lipid compositionaccording to any one of the twentieth through twentyseventh embodimentsfurther comprising a stealth lipid.

In a twentyninth embodiment, the invention is the lipid compositionaccording to any one of the twentieth through twentyeighth embodiments,wherein the helper lipid is cholesterol, the neutral lipid is DSPC, andthe stealth lipid is PEG-DMG, S010, S011 or S024.

In a thirtieth embodiment, the invention is the lipid compositionaccording to any one of the twentieth through twentyninth embodiments,wherein the lipid composition is in the form of a lipid nanoparticle.

In a thirtyfirst embodiment, the invention is the lipid compositionaccording to any one of the twentieth through thirtieth embodiments,having 30-60% of a compound of formula (I), 5-10% cholesterol/30-60%DSPC, and 0.1-5% PEG-DMG, S010, S011 or S024

In a thirtysecond embodiment, the invention is the lipid compositionaccording to any one of the twentieth through thirtyfirst embodiments,wherein the pH of said lipid composition is 4-6 at the time ofencapsulation and/or formulation.

In a thirtythird embodiment, the invention is the lipid compositionaccording to any one of the twentieth through thirtysecond embodiments,wherein the pH of said lipid composition is 5-6 at the time ofencapsulation and/or formulation.

In a thirtyfourth embodiment, the invention is the lipid compositionaccording to any one of the twentieth through thirtythird embodiments,wherein the pH of said lipid composition is 5.6-6.0 at the time ofencapsulation and/or formulation.

In a thirtyfifth embodiment, the invention is a pharmaceuticalcomposition comprising a lipid composition according to any one of thetwentieth through thirtyfourth embodiments and a pharmaceuticallyacceptable carrier or excipient.

In a thirtysixth embodiment, the invention is a method for the treatmentof a disease or condition comprising the step of administering atherapeutically effective amount of lipid composition according to anyone of the twentieth through thirtyfifth embodiments to a patient inneed of treatment thereof.

In a thirtyseventh embodiment, the invention is a method for thetreatment of a disease or condition comprising the step of administeringa therapeutically effective amount of pharmaceutical compositionaccording to the thirtysixth embodiment.

In a thirtyeighth embodiment, the invention is the composition of anyone of the twentieth through thirtyfifth embodiment, wherein thecomposition comprises a RNA molecule that encodes an immunogen.

In a thirtyninth embodiment, the invention is the composition of thethirtyeighth embodiment, wherein the lipid is in the form of a lipidnanoparticle (LNP) and the RNA is associated with the LNP.

In a fortieth embodiment, the invention is the composition ofthirtyninth embodiment wherein the LNP is a liposome.

In a fortyfirst embodiment, the invention is the composition of of thefortieth embodiment, wherein the liposome has a diameter in the range ofabout: 60-180 nm, e.g., about: 80-160 nm.

In a fortysecond embodiment, the invention is the composition of thefortieth or fortyfist embodiment, wherein the liposome is is about:80-160 nm and wherein at least half of the molar percentage of the RNAmolecules are encapsulated in the liposomes.

In a fortythird embodiment, the invention is the composition of any oneof the fortieth through fortysecond embodiments, wherein said liposomefurther comprises a lipid comprising a zwitterionic head group.

In a fortyfourth embodiment, the invention is the composition of any oneof the fortieth through fortythird embodiments, wherein said liposomefurther comprises DlinDMA(1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane), DSPC(1,2-Diastearoyl-sn-glycero-3-phosphocholine), a cholesterol, aPEGylated lipid, or a combination thereof.

In a fortyfifth embodiment, the invention is a pharmaceuticalcomposition comprising the composition of any one of the fortieththrough fortyfourth embodiments and a pharmaceutical excipient.

In a fortysixth embodiment, the invention is a pharmaceuticalcomposition comprising liposomes and immunogen-encoding RNA molecules,wherein the liposomes comprise the compound of any one of the firstthrough nineteenth embodiments, and wherein at least half of the molarpercentage of the RNA molecules are encapsulated in the liposomes.

In a fortyseventh embodiment, the invention is the pharmaceuticalcomposition of the fortysixth embodiment, wherein (i) at least 80% bynumber of the liposomes have diameters in the range of about: 60-180 nm,(ii) the average diameter of the liposomes is in the range of about:60-180 nm, or (iii) the diameters of the liposomes have a polydispersityindex of <0.2.

In a fortyeighth embodiment, the invention is the composition of any oneof the fortieth through fortyseventh embodiments, wherein the RNA is aself-replicating RNA.

In a fortyninth embodiment, the invention is the composition of of thefortyeighth embodiment, wherein the self-replicating RNA encodes aRNA-dependent RNA polymerase.

In a fiftieth embodiment, the invention is the composition of thefortyeighth or fortyninth embodiments, wherein the self-replicating RNAcomprises a first open reading frame that encodes an alphavirusreplicase and a second open reading frame that encodes the immunogen.

In a fiftyfirst embodiment, the invention is the composition of any oneof the fortyeighth through fiftieth embodiments, wherein theself-replicating RNA is greater than about 2000 nucleotides, such asgreater than about: 9000, 12000, 15000, 18000, 21000, 24000, or morenucleotides long.

In a fiftysecond embodiment, the invention is the composition of any oneof the thirtyeighth through fiftyfirst embodiments, wherein theimmunogen can elicit an immune response in vivo against a bacterium, avirus, a fungus or a parasite.

In a fiftythird embodiment, the invention is a method for inducing animmune response to an immunogen in a vertebrate, comprisingadministering an effective amount of the composition of any one of thethirtyeighth through fiftysecond embodiments to the vertebrate.

In a fiftyfourth embodiment, the invention is the composition of any oneof the thirtyeighth through fiftysecond embodiments for inducing animmune response in a vertebrate to an immunogen.

As used herein, the term “alkyl” refers to a fully saturated branched orunbranched hydrocarbon chain having the specified number of carbonatoms. For example, C₁₋₈ alkyl refers to an alkyl group having from 1 to8 carbon atoms. For example, C₄₋₂₂ alkyl refers to an alkyl group havingfrom 4 to 22 carbon atoms. For example, C₆₋₁₀ alkyl refers to an alkylgroup having from 6 to 10 carbon atoms. For example, C₁₂₋₂₂ alkyl refersto an alkyl group having from 12 to 22 carbon atoms. Representativeexamples of alkyl include, but are not limited to, methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl,n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl,n-decyl, n-undecanyl, n-dodecanyl, n-tridecanyl, 9-methylheptadecanyl,1-heptyldecyl, 2-octyldecyl, 6-hexyldodecyl, 4-heptylundecyl, and thelike.

As used herein, the term “alkylene” refers to divalent alkyl group asdefined herein above. Representative examples of alkylene include, butare not limited to, methylene, ethylene, n-propylene, iso-propylene,n-butylene, sec-butylene, iso-butylene, tert-butylene, n-pentylene,isopentylene, neopentylene, n-hexylene, 3-methylhexylene,2,2-dimethylpentylene, 2,3-dimethylpentylene, n-heptylene, n-octylene,n-nonylene, n-decylene, and the like.

As used herein, the term “alkenyl” refers to an unsaturated branched orunbranched hydrocarbon chain having the specified number of carbon atomsand one or more carbon-carbon double bonds within the chain. Forexample, C₁₂₋₂₂ alkenyl refers to an alkenyl group having 12 to 22carbon atoms with one or more carbon-carbon double bonds within thechain. In certain embodiments alkenyl groups have one carbon-carbondouble bond within the chain. In other embodiments, alkenyl groups havemore than one carbon-carbon double bond within the chain. Alkyenylgroups may be optionally substituted with one or more substituents asdefined in formula (I). Representative examples of alkenyl include, butare not limited to, ethylenyl, propenyl, butenyl, pentenyl, hexenyl andthe like. Other examples of alkenyl include, but are not limited to:Z-octadec-9-enyl, Z-undec-7-enyl, Z-heptadeca-8-enyl,(9Z,12Z)-octadeca-9,12-dienyl, (8Z,11Z)-heptadeca-8,11-dienyl, (8Z, 11Z,14Z)-heptadeca-8,11,14-trienyl, linolenyl, 2-octyldeca-1-enyl, linoleyland olelyl.

As used herein, the term “alkenylene” refers a divalent alkenyl group asdefined herein above. Representative examples of alkenylene include, butare not limited to, ethenylene, propenylene, butenylene, pentenylene,hexenylene and the like.

As used herein, the term “alkoxy” refers to refers to any alkyl moietyattached through an oxygen bridge (i.e. a —O—C₁₋₃ alkyl group whereinC₁₋₃ alkyl is as defined herein). Examples of such groups include, butare not limited to, methoxy, ethoxy, and propoxy.

As used herein, the term “cycloalkyl” refers to a saturated monocyclic,bicyclic or tricyclic hydrocarbon ring having the specified number ofcarbon atoms. For example, C₃₋₇ cycloalkyl refers to a cycloalkyl ringhaving from 3 to 7 carbon atoms. Cycloalkyl groups may be optionallysubstituted with one or more substituents as defined in formula (I).Representative examples of cycloalkyl include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.1.1]hexyl,bicyclo[2.2.1]heptyl, adamantyl and the like.

As used herein, the term “halo” refers to fluoro, chloro, bromo, andiodo.

As used herein, the term “heterocyclic” refers to a 4 to 12 memberedsaturated or unsaturated monocyclic or bicyclic ring containing from 1to 4 heteroatoms. Heterocyclic ring systems are not aromatic.Heterocyclic groups containing more than one heteroatom may containdifferent heteroatoms. Heterocyclic groups are monocyclic, spiro, orfused or bridged bicyclic ring systems. Examples of monocyclicheterocyclic groups include tetrahydrofuranyl, dihydrofuranyl,1,4-dioxanyl, morpholinyl, 1,4-dithianyl, azetidinyl, piperazinyl,piperidinyl, 1,3-dioxolanyl, imidazolidinyl, imidazolinyl, pyrrolinyl,pyrrolidinyl, tetrahydropyranyl, dihydropyranyl,1,2,3,6-tetrahydropyridinyl, oxathiolanyl, dithiolanyl, 1,3-dioxanyl,1,3-dithianyl, oxathianyl, thiomorpholinyl,1,4,7-trioxa-10-azacyclododecanyl, azapanyl and the like. Examples ofspiro heterocyclic rings include, but are not limited to,1,5-dioxa-9-azaspiro[5.5]undecanyl, 1,4-dioxa-8-azaspiro[4.5]decanyl,2-oxa-7-azaspiro[3.5]nonanyl, and the like. Fused heterocyclic ringsystems have from 8 to 11 ring atoms and include groups wherein aheterocyclic ring is fused to a phenyl ring. Examples of fusedheterocyclic rings include, but are not limited to decahydroqunilinyl,(4aS,8aR)-decahydroisoquinolinyl, (4aS,8aS)-decahydroisoquinolinyl,octahydrocyclopenta[c]pyrrolyl, isoinolinyl,(3aR,7aS)-hexahydro-[1,3]dioxolo[4.5-c]pyridinyl,octahydro-1H-pyrrolo[3,4-b]pyridinyl, tetrahydroisoquinolinyl and thelike.

As used herein, the term “heterocyclylC₁₋₈alkyl” refers to aheterocyclic ring as defined above which is attached to the rest of themolecule by a single bond or by a C₁₋₈alkyl radical as defined above.

As used herein, the term “heteroaryl” refers to a 5- or 6-memberedaromatic monocyclic ring radical which comprises 1, 2, 3 or 4heteroatoms individually selected from nitrogen, oxygen and sulfur. Theheteroaryl radical may be bonded via a carbon atom or heteroatom.Examples of heteroaryl include, but are not limited to, furyl, pyrrolyl,thienyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl,isoxazolyl, triazolyl, tetrazolyl, pyrazinyl, pyridazinyl, pyrimidyl orpyridyl.

As used herein, the term “heteroarylC₁₋₈alkyl” refers to a heteroarylring as defined above which is attached to the rest of the molecule by asingle bond or by a C₁₋₈alkyl radical as defined above. As used herein,the term “an optical isomer” or “a stereoisomer” refers to any of thevarious stereoisomeric configurations which may exist for a givencompound of the present invention and includes geometric isomers. It isunderstood that a substituent may be attached at a chiral center of acarbon atom. The term “chiral” refers to molecules which have theproperty of non-superimposability on their mirror image partner, whilethe term “achiral” refers to molecules which are superimposable on theirmirror image partner. Therefore, the invention includes enantiomers,diastereomers or racemates of the compound. “Enantiomers” are a pair ofstereoisomers that are non-superimposable mirror images of each other. A1:1 mixture of a pair of enantiomers is a “racemic” mixture. The term isused to designate a racemic mixture where appropriate.“Diastereoisomers” are stereoisomers that have at least two asymmetricatoms, but which are not mirror-images of each other. The absolutestereochemistry is specified according to the Cahn-Ingold-Prelog R-Ssystem. When a compound is a pure enantiomer the stereochemistry at eachchiral carbon may be specified by either R or S. Resolved compoundswhose absolute configuration is unknown can be designated (+) or (−)depending on the direction (dextro- or levorotatory) which they rotateplane polarized light at the wavelength of the sodium D line. Certaincompounds described herein contain one or more asymmetric centers oraxes and may thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)-.

Depending on the choice of the starting materials and procedures, thecompounds can be present in the form of one of the possible isomers oras mixtures thereof, for example as pure optical isomers, or as isomermixtures, such as racemates and diastereoisomer mixtures, depending onthe number of asymmetric carbon atoms. The present invention is meant toinclude all such possible isomers, including racemic mixtures,diasteriomeric mixtures and optically pure forms. Optically active (R)-and (S)-isomers may be prepared using chiral synthons or chiralreagents, or resolved using conventional techniques. If the compoundcontains a double bond, the substituent may be E or Z configuration. Ifthe compound contains a disubstituted cycloalkyl, the cycloalkylsubstituent may have a cis- or trans-configuration. All tautomeric formsare also intended to be included.

Any asymmetric atom (e.g., carbon or the like) of the compound(s) of thepresent invention can be present in racemic or enantiomericallyenriched, for example the (R)-, (S)- or (R,S)-configuration. In certainembodiments, each asymmetric atom has at least 50% enantiomeric excess,at least 60% enantiomeric excess, at least 70% enantiomeric excess, atleast 80% enantiomeric excess, at least 90% enantiomeric excess, atleast 95% enantiomeric excess, or at least 99% enantiomeric excess inthe (R)- or (S)-configuration. Substituents at atoms with unsaturateddouble bonds may, if possible, be present in cis-(Z)- or trans-(E)-form.

Accordingly, as used herein a compound of the present invention can bein the form of one of the possible isomers, rotamers, atropisomers,tautomers or mixtures thereof, for example, as substantially puregeometric (cis or trans) isomers, diastereomers, optical isomers(antipodes), racemates or mixtures thereof.

Any resulting mixtures of isomers can be separated on the basis of thephysicochemical differences of the constituents, into the pure orsubstantially pure geometric or optical isomers, diastereomers,racemates, for example, by chromatography and/or fractionalcrystallization.

Any resulting racemates of final products or intermediates can beresolved into the optical antipodes by known methods, e.g., byseparation of the diastereomeric salts thereof, obtained with anoptically active acid or base, and liberating the optically activeacidic or basic compound. In particular, a basic moiety may thus beemployed to resolve the compounds of the present invention into theiroptical antipodes, e.g., by fractional crystallization of a salt formedwith an optically active acid, e.g., tartaric acid, dibenzoyl tartaricacid, diacetyl tartaric acid, di-O,O′-p-toluoyl tartaric acid, mandelicacid, malic acid or camphor-10-sulfonic acid. Racemic products can alsobe resolved by chiral chromatography, e.g., high pressure liquidchromatography (HPLC) using a chiral adsorbent.

As used herein, the terms “salt” or “salts” refers to an acid additionof a compound of the invention. “Salts” include in particular“pharmaceutically acceptable salts”. The term “pharmaceuticallyacceptable salts” refers to salts that retain the biologicaleffectiveness and properties of the compounds of this invention and,which typically are not biologically or otherwise undesirable. In manycases, the compounds of the present invention are capable of formingacid and/or base salts by virtue of the presence of amino and/orcarboxyl groups or groups similar thereto.

Pharmaceutically acceptable acid addition salts can be formed withinorganic acids and organic acids, e.g., acetate, aspartate, benzoate,besylate, bromide/hydrobromide, bicarbonate/carbonate,bisulfate/sulfate, camphorsulfonate, chloride/hydrochloride,chlortheophyllonate, citrate, ethandisulfonate, fumarate, gluceptate,gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate,lactate, lactobionate, laurylsulfate, malate, maleate, malonate,mandelate, mesylate, methylsulphate, naphthoate, napsylate, nicotinate,nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate,phosphate/hydrogen phosphate/dihydrogen phosphate, polygalacturonate,propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate andtrifluoroacetate salts.

Inorganic acids from which salts can be derived include, for example,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like.

Organic acids from which salts can be derived include, for example,acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,toluenesulfonic acid, sulfosalicylic acid, and the like.Pharmaceutically acceptable base addition salts can be formed withinorganic and organic bases.

Inorganic bases from which salts can be derived include, for example,ammonium salts and metals from columns I to XII of the periodic table.In certain embodiments, the salts are derived from sodium, potassium,ammonium, calcium, magnesium, iron, silver, zinc, and copper;particularly suitable salts include ammonium, potassium, sodium, calciumand magnesium salts.

Organic bases from which salts can be derived include, for example,primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, basic ionexchange resins, and the like. Certain organic amines includeisopropylamine, benzathine, cholinate, diethanolamine, diethylamine,lysine, meglumine, piperazine and tromethamine.

The pharmaceutically acceptable salts of the present invention can besynthesized from a basic or acidic moiety, by conventional chemicalmethods. Generally, such salts can be prepared by reacting free acidforms of these compounds with a stoichiometric amount of the appropriatebase (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate or thelike), or by reacting free base forms of these compounds with astoichiometric amount of the appropriate acid. Such reactions aretypically carried out in water or in an organic solvent, or in a mixtureof the two. Generally, use of non-aqueous media like ether, ethylacetate, ethanol, isopropanol, or acetonitrile is desirable, wherepracticable. Lists of additional suitable salts can be found, e.g., in“Remington's Pharmaceutical Sciences”, 20th ed., Mack PublishingCompany, Easton, Pa., (1985); and in “Handbook of Pharmaceutical Salts:Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH,Weinheim, Germany, 2002).

General Methods for Synthesizing Cationic Lipids

The present invention also includes processes for the preparation ofcompounds of formula (I). In the reactions described, it could benecessary to protect reactive functional groups, for example hydroxyl,amino, iminio, thio or carboxy groups, where these are desired in thefinal product, to avoid their unwanted participation in the reactions.

The compounds and processes of the present invention will be betterunderstood in connection with the following synthetic schemes, which aremerely intended to illustrate methods by which the compounds may begenerally prepared and are not intended to limit the scope of theinvention as defined in the claims.

Final compounds of formula (I) can be prepared as described in FIG. 1.

Lipid Compositions

The present invention provides for a lipid composition comprising atleast one compound of formula (I), i.e. a lipid composition of theinvention. In one embodiment, at least one other lipid component ispresent. Such compositions can also contain a biologically active agent,optionally in combination with one or more other lipid components.

One embodiment of the present invention provides for a lipid compositioncomprising a compound of formula (I) and another lipid component. Suchother lipid components include, but are not limited to, cationic lipids,neutral lipids, anionic lipids, helper lipids, and stealth lipids.

Cationic lipids suitable for use in a lipid composition of the inventioninclude, but are not limited to, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy) propyl)-N,N,N-trimethylammonium chloride (DOTAP),1,2-Dioleoyl-3-Dimethylammonium-propane (DODAP),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),1,2-Dioleoylcarbamyl-3-Dimethylammonium-propane (DOCDAP),1,2-Dilineoyl-3-Dimethylammonium-propane (DLINDAP), dilauryl(C_(12:0))trimethyl ammonium propane (DLTAP), Dioctadecylamidoglycyl spermine(DOGS), DC-Chol,Dioleoyloxy-N-[2-sperminecarboxamido)ethyl}-N,N-dimethyl-1-propanaminiumtrifluoroacetate(DOSPA), 1,2-Dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammoniumbromide (DMRIE),3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),2-[5′-(cholest-5-en-3[beta]-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane (CpLinDMA) and N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA),and 1,2-N,N′-Dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP). In oneembodiment the cationic lipid is DOTAP or DLTAP.

“Neutral lipids” suitable for use in a lipid composition of theinvention include, for example, a variety of neutral, uncharged orzwitterionic lipids. Examples of neutral phospholipids suitable for usein the present invention include, but are not limited to:5-heptadecylbenzene-1,3-diol (resorcinol),dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC),phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine(DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC),dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine(DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC),I-palmitoyl-2-myristoyl phosphatidylcholine (PMPC),1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC),1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC),1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC),1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidyl choline, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholinedistearoylphophatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine(DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE),lysophosphatidylethanolamine and combinations thereof. In oneembodiment, the neutral phospholipid is selected from the groupconsisting of distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidyl ethanolamine (DMPE).

Anionic lipids suitable for use in the present invention include, butare not limited to, phosphatidylglycerol, cardiolipin,diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidyl ethanoloamine, N-succinyl phosphatidylethanolamine,N-glutaryl phosphatidylethanolamine cholesterol hemisuccinate (CHEMS),and lysylphosphatidylglycerol.

Suitable neutral and anionic lipids also include those described in US2009/0048197.

“Helper lipids” are lipids that enhance transfection (e.g. transfectionof the nanoparticle including the biologically active agent) to someextent. The mechanism by which the helper lipid enhances transfectionmay include, e.g., enhancing particle stability and/or enhancingmembrane fusogenicity. Helper lipids include steroids and alkylresorcinols. Helper lipids suitable for use in the present inventioninclude, but are not limited to, cholesterol, 5-heptadecylresorcinol,and cholesterol hemisuccinate.

Stealth lipids are lipids that increase the length of time for which thenanoparticles can exist in vivo (e.g. in the blood). Stealth lipidssuitable for use in a lipid composition of the invention include, butare not limited to, stealth lipids having a hydrophilic head grouplinked to a lipid moiety. Examples of such stealth lipids includecompounds of formula (XI), as described in WO2011/076807,

or a salt or pharmaceutically acceptable derivative thereof,wherein:

Z_(n) is a hydrophilic polymer moiety selected from PEGpoly(ethyleneoxide) or polymers based on poly(oxazoline), poly(vinylalcohol), poly(glycerol), poly(N-vinylpyrro-lidone),poly[N-(2-hydroxypropyl)methacrylamide], polysaccharides and poly(aminoacid)s or a combination of the foregoing, wherein the polymer may belinear or branched, and wherein each Z is independently and optionallymay be optionally substituted;

wherein Z is polymerized by n subunits;

n is a number-averaged degree of polymerization between 10 and 200 unitsof Z, wherein n is optimized for different polymer types;

L₁ is an optionally substituted C₁₋₁₀ alkylene or C₁₋₁₀ heteroalkylenelinker including zero, one, two or more of an ether (e.g., —O—), ester(e.g., —C(O)O—), succinate (e.g., —O(O)C—CH₂—CH₂—C(O)O—)), carbamate(e.g., —OC(O)—NR′—), carbonate (e.g., —OC(O)O—), ketone (e.g.,—C—C(O)—C—), carbonyl (e.g., —C(O)—), urea (e.g., —NRC(O)NR′—), amine(e.g., —NR′—), amide (e.g., —C(O)NR′—), imine (e.g., —C(NR′)—),thioether (e.g., —S—), xanthate (e.g., —OC(S)S—), and phosphodiester(e.g., —OP(O)₂O—); any of which may be substituted by zero, one or moreZ groups;

wherein R′ is independently selected from —H, —NH—, —NH₂, —O—, —S—, aphosphate or an optionally substituted C₁₋₁₀ alkylene;

X₁ and X₂ are independently selected from a carbon or a heteroatomselected from —NH—, —O—, —S— or a phosphate;

A₁ and A₂ are independently selected from a C₆₋₃₀ alkyl, C₆₋₃₀ alkenyl,and C₆₋₃₀ alkynyl, wherein A₁ and A₂ may be the same or different,

or wherein A₁ and A₂ together with the carbon atom to which they areattached form an optionally substituted steroid.

Specific stealth lipids include, but are not limited to, those listed inTable 1.

TABLE 1 Stealth Lipids Stealth Lipid Lipid S001

S002

S003

S004

S005

S006

S007

S008

S009

S010

S011

S012

S013

S014

S015

S016

S017

S018

S019

S020

S021

S022

S023

S024

S025

S026

S027

S028

S029

S030

S031

S032

S033

Other stealth lipids suitable for use in a lipid composition of thepresent invention and information about the biochemistry of such lipidscan be found in Romberg et al., Pharmaceutical Research, Vol. 25, No. 1,2008, p. 55-71 and Hoekstra et al., Biochimica et Biophysica Acta 1660(2004) 41-52.

In one embodiment, the suitable stealth lipid comprises a group selectedfrom PEG (sometimes referred to as poly(ethylene oxide) and polymersbased on poly(oxazoline), poly(vinyl alcohol), poly(glycerol),poly(N-vinylpyrrolidone), polyaminoacids and poly[N-(2-hydroxypropyl)methacrylamide]. Additional suitable PEG lipids are disclosed, e.g., inWO 2006/007712.

Specific suitable stealth lipids includepolyethyleneglycol-diacylglycerol or polyethyleneglycol-diacylglycamide(PEG-DAG) conjugates including those comprising a dialkylglycerol ordialkylglycamide group having alkyl chain length independentlycomprising from about C₄ to about C₄₀ saturated or unsaturated carbonatoms. The dialkylglycerol or dialkylglycamide group can furthercomprise one or more substituted alkyl groups. In any of the embodimentsdescribed herein, the PEG conjugate can be selected fromPEG-dilaurylglycerol, PEG-dimyristylglycerol (PEG-DMG) (catalog # GM-020from NOF, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG-disterylglycerol,PEG-dilaurylglycamide, PEG-dimyristylglycamide,PEG-dipalmitoylglycamide, and PEG-disterylglycamide, PEG-cholesterol(1-[8′-(Cholest-5-en-3[beta]-oxy)carboxamido-3′,6′-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethyleneglycol), PEG-DMB (3,4-Ditetradecoxylbenzyl-[omega]-methyl-poly(ethyleneglycol) ether),1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (catalog #880150P from Avanti Polar Lipids, Alabaster,Ala., USA).

In one embodiment the stealth lipid is S010, S024, S027, S031, or S033.

In another embodiment the stealth lipid is S024.

Unless otherwise indicated, the term “PEG” as used herein means anypolyethylene glycol or other polyalkylene ether polymer. In oneembodiment, PEG is an optionally substituted linear or branched polymerof ethylene glycol or ethylene oxide. In one embodiment PEG isunsubstituted. In one embodiment the PEG is substituted, e.g., by one ormore alkyl, alkoxy, acyl, hydroxy or aryl groups. In one embodiment, theterm includes PEG copolymers such as PEG-polyurethane orPEG-polypropylene (see, e.g., J. Milton Harris, Poly(ethylene glycol)chemistry: biotechnical and biomedical applications (1992)); in anotherembodiment, the term does not include PEG copolymers. In one embodiment,the PEG has a molecular weight of from about 130 to about 50,000, in asub-embodiment about 150 to about 30,000, in a sub-embodiment about 150to about 20,000, in a sub-embodiment about 150 to about 15,000, in asub-embodiment about 150 to about 10,000, in a sub-embodiment about 150to about 6000, in a sub-embodiment about 150 to about 5000, in asub-embodiment about 150 to about 4000, in a sub-embodiment about 150 toabout 3000, in a sub-embodiment about 300 to about 3000, in asub-embodiment about 1000 to about 3000, and in a sub-embodiment about1500 to about 2500.

In certain embodiments the PEG (e.g., conjugated to a lipid, such as astealth lipid) is a “PEG-2K”, also termed “PEG 2000”, which has anaverage molecular weight of about 2000 daltons. PEG-2K is representedherein by the following formula (XIIa), wherein n is 45, meaning thatthe number-averaged degree of polymerization comprises about 45subunits. However, other PEG embodiments known in the art may be used,including, e.g., those where the number-averaged degree ofpolymerization comprises about 23 subunits (n=23) and/or 68 subunits(n=68).

The lipid compositions of the invention can also include one or morebiologically active agents including, but not limited to, antibodies(e.g., monoclonal, chimeric, humanized, nanobodies, and fragmentsthereof etc.), cholesterol, hormones, peptides, proteins,chemotherapeutics and other types of antineoplastic agents, lowmolecular weight drugs, vitamins, co-factors, nucleosides, nucleotides,oligonucleotides, enzymatic nucleic acids, antisense nucleic acids,triplex forming oligonucleotides, antisense DNA or RNA compositions,chimeric DNA:RNA compositions, allozymes, aptamers, ribozyme, decoys andanalogs thereof, plasmids and other types of expression vectors, andsmall nucleic acid molecules, RNAi agents, short interfering nucleicacid (siNA), messenger ribonucleic acid” (messenger RNA, mRNA), shortinterfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA),and short hairpin RNA (shRNA) molecules, peptide nucleic acid (PNA), alocked nucleic acid ribonucleotide (LNA), morpholino nucleotide, threosenucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (small internallysegmented interfering RNA), aiRNA (assymetrical interfering RNA), andsiRNA with 1, 2 or more mismatches between the sense and anti-sensestrand to relevant cells and/or tissues, such as in a cell culture,subject or organism. Such compounds may be purified or partiallypurified, and may be naturally occurring or synthetic, and may bechemically modified. In one embodiment the biologically active agent isan RNAi agent, short interfering nucleic acid (siNA), short interferingRNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), or a shorthairpin RNA (shRNA) molecule. In one embodiment the biologically activeagent is a RNAi agent useful for mediating RNA interference (RNAi). Inanother embodiment the biologically active agent is a mRNA.

Various methods for loading biologically active agents into lipidcompositions, such as liposomes and lipid nanoparticles are available inthe art, including both passive and active loading methods. The exactmethod used may be chosen based on multiple factors that include, butare not limited to, e.g., the biologically active agent to be loaded,the storage method to be used once loaded, the size of the resultingparticle, and the dosage regimen contemplated. Methods include, e.g.,mechanical mixing of the drug and lipids at the time the liposomes areformed or reconstituted, dissolving all components in an organic solventand concentrating them into a dry film, forming a pH or ion gradient todraw the active agent into the interior of the liposome, creating atransmembrane potential, and ionophore mediated loading. See, e.g., PCTPublication No. WO 95/08986, U.S. Pat. Nos. 5,837,282, 5,837,282, and7,811,602.

By “lipid nanoparticle” is meant a particle that comprises a pluralityof (i.e. more than one) lipid molecules physically associated with eachother by intermolecular forces. The lipid nanoparticles may be, e.g.,microspheres (including unilamellar and multlamellar vesicles, e.g.“liposomes”—lamellar phase libid bilayers that, in some embodiments aresubstantially spherical, and, in more particular embodiments cancomprise an aqueous core, e.g., comprising a substantial portion of RNAmolecules), a dispersed phase in an emulsion, micelles or an internalphase in a suspension.

The lipid nanoparticles have a size of about 1 to about 2,500 nm, about10 to about 1,500 nm, about 20 to about 1,000 nm, in a sub-embodimentabout 50 to about 600 nm, in a sub-embodiment about 50 to about 400 nm,in a sub-embodiment about 50 to about 250 nm, and in a sub-embodimentabout 50 to about 150 nm. Unless indicated otherwise, all sizes referredto herein are the average sizes (diameters) of the fully formednanoparticle, as measured by dynamic light scattering on a MalvernZetasizer. The nanoparticle sample is diluted in phosphate bufferedsaline (PBS) so that the count rate is approximately 200-400 kcts. Thedata is presented as a weighted average of the intensity measure.

One embodiment of the present invention provides for a lipid compositioncomprising a compound of formula (I) and another lipid component.Another embodiment provides for a lipid composition comprising acompound of formula (I) and a helper lipid, for example cholesterol.Another embodiment provides for a lipid composition comprising acompound of formula (I), a helper lipid, for example cholesterol, and aneutral lipid, for example DSPC. Another embodiment of the presentinvention provides for a lipid composition comprising a compound offormula (I), a helper lipid, for example cholesterol, a neutral lipid,for example DSPC, and a stealth lipid, for example S010, S024, S027,S031, or S033. Another embodiment of the present invention provides fora lipid composition comprising a compound of formula (I), a helperlipid, for example cholesterol, a neutral lipid, for example DSPC, astealth lipid, for example S010, S024, S027, S031, or S033, and abiologically active agent, for example a RNA or DNA. Another embodimentof the present invention provides for a lipid nanoparticle comprising acompound of formula (I) a helper lipid, for example cholesterol, aneutral lipid, for example DSPC, and a stealth lipid, for example S010,S024, S027, S031, or S033, and a biologically active agent, for examplea mRNA, siRNA or DNA.

Embodiments of the present invention also provide lipid compositionsdescribed according to the respective molar ratios of the componentlipids in the formulation, wherein a slash (“/”) indicates therespective components, as provided herein.

Another embodiment of the present invention is a lipid compositioncomprising a compound of formula (I) and a helper lipid, for examplecholesterol, in a lipid molar ratio of 55-40 compound of formula(I)/55-40 helper lipid. Another embodiment provides for a lipidcomposition comprising a compound of formula (I), a helper lipid, forexample cholesterol, and a neutral lipid, for example DPSC in a lipidmolar ratio of 55-40 compound of formula (I)/55-40 helper lipid/15-5neutral lipid. Another embodiment provides for a lipid compositioncomprising a compound of formula (I), a helper lipid, for examplecholesterol, a neutral lipid, for example DSPC, and a stealth lipid, forexample S010, S024, S027, S031, or S033 in a lipid molar ratio of 55-40compound of formula (I)/55-40 helper lipid/15-5 neutral lipid/10-1stealth lipid.

Another embodiment of the present invention is a lipid compositioncomprising a compound of formula (I) and a helper lipid, for examplecholesterol, in a lipid molar ratio of 50-40 compound of formula(I)/50-40 helper lipid. Another embodiment provides for a lipidcomposition comprising a compound of formula (I), a helper lipid, forexample cholesterol, and a neutral lipid, for example DPSC in a lipidmolar ratio of 50-40 compound of formula (I)/50-40 helper lipid/15-5neutral lipid. Another embodiment provides for a lipid compositioncomprising a compound of formula (I), a helper lipid, for examplecholesterol, a neutral lipid, for example DSPC, a stealth lipid, forexample S010, S024, S027, S031, or S033 in a lipid molar ratio of 50-40compound of formula (I)/50-40 helper lipid/15-5 neutral lipid/5-1stealth lipid.

Another embodiment of the present invention is a lipid compositioncomprising a compound of formula (I) and a helper lipid, for examplecholesterol, in a lipid molar ratio of 47-43 compound of formula(I)/47-43 helper lipid. Another embodiment provides for a lipidcomposition comprising a compound of formula (I), a helper lipid, forexample cholesterol, and a neutral lipid, for example DPSC in a lipidmolar ratio of 47-43 compound of formula (I)/47-43 helper lipid/12-7neutral lipid. Another embodiment provides for a lipid compositioncomprising a compound of formula (I), a helper lipid, for examplecholesterol, a neutral lipid, for example DSPC, a stealth lipid, forexample S010, S024, S027, S031, or S033 in a lipid molar ratio of 47-43compound of formula (I)/47-43 helper lipid/12-7 neutral lipid/4-1stealth lipid.

Another embodiment of the present invention is a lipid compositioncomprising a compound of formula (I) and a helper lipid, for examplecholesterol, in a lipid molar ratio of about 45 compound of formula(I)/about 44 helper lipid. Another embodiment provides for a lipidcomposition comprising a compound of formula (I), a helper lipid, forexample cholesterol, and a neutral lipid, for example DPSC in a lipidmolar ratio of about 45 compound of formula (I)/about 44 helperlipid/about 9 neutral lipid. Another embodiment provides for a lipidcomposition comprising a compound of formula (I), a helper lipid, forexample cholesterol, a neutral lipid, for example DSPC, a stealth lipid,for example S010, S024, S027, S031, or S033 in a lipid molar ratio ofabout 45 compound of formula (I)/about 44 helper lipid/about 9 neutrallipid/about 2 stealth lipid, for example S010, S024, S027, S031, orS033.

Preferred compounds of formula (I) for use in the above lipidcompositions are given in Examples 1-36. Particularly preferredcompounds are given in Examples 1 and 80. Preferred biologically activeagents are RNA's and DNA's.

Lipid compositions of the present invention can be further optimized byone skilled in the art by combining cationic lipids with the desired pKarange, stealth lipids, helper lipids, and neutral lipids intoformulations, including, e.g., liposome formulations, lipidnanoparticles (LNP) formulations, and the like for delivery to specificcells and tissues in vivo. In one embodiment, further optimization isobtained by adjusting the lipid molar ratio between these various typesof lipids. In one embodiment, further optimization is obtained byadjusting one or more of: the desired particle size, N/P ratio,formulation methods and/or dosing regimen (e.g., number of dosesadministered over time, actual dose in mg/kg, timing of the doses,combinations with other therapeutics, etc.). The various optimizationtechniques known to those of skill in the art pertaining to the abovelisted embodiments are considered as part of this invention.

General Methods for Making Lipid Nanoparticles

The following methods can be used to make lipid nanoparticles of theinvention. To achieve size reduction and/or to increase the homogeneityof size in the particles, the skilled person may use the method stepsset out below, experimenting with different combinations. Additionally,the skilled person could employ sonication, filtration or other sizingtechniques which are used in liposomal formulations.

The process for making a composition of the invention typicallycomprises providing an aqueous solution, such as citrate buffer,comprising a biologically active agent in a first reservoir, providing asecond reservoir comprising an organic solution, such as an organicalcohol, for example ethanol, of the lipid(s) and then mixing theaqueous solution with the organic lipid solution. The first reservoir isoptionally in fluid communication with the second reservoir. The mixingstep is optionally followed by an incubation step, a filtration ordialysis step, and a dilution and/or concentration step. The incubationstep comprises allowing the solution from the mixing step to stand in avessel for about 0 to about 100 hours (preferably about 0 to about 24hours) at about room temperature and optionally protected from light. Incertain embodiments, the temperature may be about 4° C. duringincubation. In one embodiment, a dilution step follows the incubationstep. The dilution step may involve dilution with aqueous buffer (e.g.citrate buffer or pure water) e.g., using a pumping apparatus (e.g. aperistaltic pump). The filtration step is ultrafiltration or dialysis.Ultrafiltration comprises concentration of the diluted solution followedby diafiltration, e.g., using a suitable pumping system (e.g. pumpingapparatus such as a peristaltic pump or equivalent thereof) inconjunction with a suitable ultrafiltration membrane (e.g. GE Hollowfiber cartridges or equivalent). Dialysis comprises solvent (buffer)exchange through a suitable membrane (e.g. 10,000 mwc snakeskinmembrane). Alternatively, in some embodiments, dialysis may beaccomplished using buffer exchange columns (e.g., PD-10 columns from GEhealthcare).

In one embodiment, the mixing step provides a clear single phase.

In one embodiment, after the mixing step, the organic solvent is removedto provide a suspension of particles, wherein the biologically activeagent is encapsulated by the lipid(s).

The selection of an organic solvent will typically involve considerationof solvent polarity and the ease with which the solvent can be removedat the later stages of particle formation. The organic solvent, which isalso used as a solubilizing agent, is preferably in an amount sufficientto provide a clear single phase mixture of biologically active agentsand lipids. The organic solvent may be selected from one or more (e.g.two) of chloroform, dichloromethane, diethylether, cyclohexane,cyclopentane, benzene, toluene, methanol, and other aliphatic alcohols(e.g. C₁ to C₈) such as ethanol, propanol, isopropanol, butanol,tert-butanol, iso-butanol, pentanol and hexanol.

The mixing step can take place by any number of methods, e.g., bymechanical means such as a vortex mixer.

The methods used to remove the organic solvent will typically involvediafilitration or dialysis or evaporation at reduced pressures orblowing a stream of inert gas (e.g. nitrogen or argon) across themixture.

In other embodiments, the method further comprises adding nonlipidpolycations which are useful to effect the transformation of cells usingthe present compositions. Examples of suitable nonlipid polycationsinclude, but are limited to, hexadimethrine bromide (sold under thebrandname POLYBRENE®, from Aldrich Chemical Co., Milwaukee, Wis., USA)or other salts of hexadimethrine. Other suitable polycations include,e.g., salts of poly-L-ornithine, poly-L-arginine, poly-L-lysine,poly-D-lysine, polyallylamine and polyethyleneimine. In certainembodiments, the formation of the lipid nanoparticles can be carried outeither in a mono-phase system (e.g. a Bligh and Dyer monophase orsimilar mixture of aqueous and organic solvents) or in a two-phasesystem with suitable mixing.

The lipid nanoparticle may be formed in a mono- or a bi-phase system. Ina mono-phase system, the cationic lipid(s) and biologically active agentare each dissolved in a volume of the mono-phase mixture. Combining thetwo solutions provides a single mixture in which the complexes form. Ina bi-phase system, the cationic lipids bind to the biologically activeagent (which is present in the aqueous phase), and “pull” it into theorganic phase. In one embodiment, the lipid nanoparticles are preparedby a method which comprises: (a) contacting the biologically activeagent with a solution comprising noncationic lipids and a detergent toform a compound-lipid mixture; (b) contacting cationic lipids with thecompound-lipid mixture to neutralize a portion of the negative charge ofthe biologically active agent and form a charge-neutralized mixture ofbiologically active agent and lipids; and (c) removing the detergentfrom the charge-neutralized mixture.

In one group of embodiments, the solution of neutral lipids anddetergent is an aqueous solution. Contacting the biologically activeagent with the solution of neutral lipids and detergent is typicallyaccomplished by mixing together a first solution of the biologicallyactive agent and a second solution of the lipids and detergent.Preferably, the biologically active agent solution is also a detergentsolution. The amount of neutral lipid which is used in the presentmethod is typically determined based on the amount of cationic lipidused, and is typically of from about 0.2 to 5 times the amount ofcationic lipid, preferably from about 0.5 to about 2 times the amount ofcationic lipid used.

The biologically active agent-lipid mixture thus formed is contactedwith cationic lipids to neutralize a portion of the negative chargewhich is associated with the molecule of interest (or other polyanionicmaterials) present. The amount of cationic lipids used is typically 3-8fold more than the calculated molar ratio of negative charge(phosphates).

The methods used to remove the detergent typically involve dialysis.When organic solvents are present, removal is typically accomplished bydiafilitration or evaporation at reduced pressures or by blowing astream of inert gas (e.g. nitrogen or argon) across the mixture.

There is herein disclosed an apparatus for making a composition of thepresent invention. The apparatus typically includes a first reservoirfor holding an aqueous solution comprising a biologically active agentand a second reservoir for holding an organic lipid solution. Theapparatus also typically includes a pump mechanism configured to pumpthe aqueous and the organic lipid solutions into a mixing region ormixing chamber at substantially equal flow rates. In one embodiment, themixing region or mixing chamber comprises a T coupling or equivalentthereof, which allows the aqueous and organic fluid streams to combineas input into the T connector and the resulting combined aqueous andorganic solutions to exit out of the T connector into a collectionreservoir or equivalent thereof. In other embodiments, a microfluidicdevice, such as a NANOASSEMBLR™, can be used for making a compositionprovided by the invention.

Methods for Delivering Biologically Active Agents and the Treatment ofDisease

The cationic lipids of formula (I) and lipid compostions thereof areuseful in pharmaceutical compositions or formulations used for deliveryof biologically active agents. Formulations containing cationic lipidsof formula (I) or lipid compositions thereof may be in various forms,including, but not limited to, particle forming delivery agentsincluding microparticles, nanoparticles and transfection agents that areuseful for delivering various molecules to cells. Specific formulationsare effective at transfecting or delivering biologically active agents,such as antibodies (e.g., monoclonal, chimeric, humanized, nanobodies,and fragments thereof etc.), cholesterol, hormones, peptides, proteins,chemotherapeutics and other types of antineoplastic agents, lowmolecular weight drugs, vitamins, co-factors, nucleosides, nucleotides,oligonucleotides, enzymatic nucleic acids, antisense nucleic acids,triplex forming oligonucleotides, antisense DNA or RNA compositions,chimeric DNA:RNA compositions, allozymes, aptamers, ribozymes, decoysand analogs thereof, plasmids and other types of expression vectors, andsmall nucleic acid molecules, mRNA, RNAi agents, short interferingnucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA(dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), and“self-replicating RNA” (encoding a replicase enzyme activity and capableof directing its own replication or amplification in vivo) molecules,peptide nucleic acid (PNA), a locked nucleic acid ribonucleotide (LNA),morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid(GNA), sisiRNA (small internally segmented interfering RNA), aiRNA(assymetrical interfering RNA), and siRNA with 1, 2 or more mismatchesbetween the sense and anti-sense strand to relevant cells and/ortissues, such as in a cell culture, subject or organism. The above listof biologically active agents is exemplary only, and is not intended tobe limiting. Such compounds may be purified or partially purified, andmay be naturally occurring or synthetic, and may be chemically modified.

Such formulations containing biologically active agents are useful,e.g., in providing compositions to prevent, inhibit, or treat diseases,conditions, or traits in a cell, subject or organism. Diseases,conditions or traits include, but are not limited to, proliferativediseases, including cancer, inflammatory disease, transplant and/ortissue rejection, autoimmune diseases or conditions, age-relateddisease, neurological or neurodegenerative disease, respiratory disease,cardiovascular disease, ocular disease, metabolic disease,dermatological disease, auditory disease, a liver disease, a kidney orrenal disease, etc.

The amount of active agent administered per dose is an amount above theminimal therapeutic dose but below a toxic dose. The actual amount perdose may be determined by a physician depending on a number of factors,such as the medical history of the patient, the use of other therapies,the biologically active agent to be provided, and the nature of thedisease. The amount of biologically active agent administered may beadjusted throughout treatment, depending on the patient's response totreatment and the presence or severity of any treatment-associated sideeffects. Exemplary dosages and treatment for compounds that have beenapproved by an appropriate regulatory agency are known and available tothose skilled in the art. See, e.g., Physician's Desk Reference, 64thed., Physician's Desk Reference Inc. (2010), Remington's PharmaceuticalSciences, Mack Publishing Company, Philadelphia, Pa. (1985), andRemington The Science and Practice of Pharmacy, 21st ed., LippincottWilliams & Williams Publishers (2005).

In one embodiment, a single dose is administered of a biologicallyactive agent to a patient in need thereof. In one embodiment, multipledoses are administered, wherein the multiple doses may be administeredconcurrently, sequentially or alternating. In one embodiment, the sameformulation is administered over multiple doses. In one embodiment, theformulations differ over multiple doses. In various embodiments, thedoses may be administered once a day, or for one, two, three, four ormore consecutive days. In one embodiment, the doses are administeredonce a week. In one embodiment, the doses are administered once everyother week. In one embodiment, patients receive at least two courses ofa treatment regimen, and potentially more, depending on the response ofthe patient to the treatment. In single agent regimens, total courses oftreatment are determined by the patient and physician based on observedresponses and toxicity. The above dosage regimens are to be consideredas non-limiting examples. Other dosage regimens are contemplated asbeing within the scope of the invention, and depend on the therapeuticeffect desired.

The invention also provides a method for the treatment of a disease orcondition comprising the step of administering a therapeuticallyeffective amount of a lipid composition of the invention to a patient inneed of treatment thereof. In one embodiment, the disease or conditionis treatable by administering a RNA agent.

The invention also provides for use of a lipid composition of theinvention in treating a disease or condition in a patient. In oneembodiment, the disease or condition is treatable by administering asiRNA or mRNA agent.

The total amount of lipid provided by the invention in the compositionbeing administered is, in one embodiment, from about 5 to about 30 mglipid per mg biologically active agent (e.g. RNA), in another embodimentfrom about 5 to about 25 mg lipid per mg biologically active agent (e.g.RNA), in another embodiment from about 7 to about 25 mg lipid per mgbiologically active agent (e.g. RNA) and in one embodiment from about 7to about 15 mg lipid per mg biologically active agent (e.g. RNA).

As used herein, “treatment” includes ameliorative, curative andprophylactic treatment. As used herein, a “patient” means an animal,preferably a mammal, preferably a human, in need of treatment.

The term “therapeutically effective amount” refers to the amount of thecompound of the invention and the biologically active agent (e.g. thetherapeutic compound) needed to treat or ameliorate a targeted diseaseor condition.

The term “immunologically effective amount” refers to the amount of thecompound of the invention and of RNA which encodes an immunogen neededto elicit an immune response which recognizes the immunogen (e.g. in thecontext of a pathogen). The term “immunogen” refers to any substance ororganism that provokes an immune response when introduced into the body.The phrase “RNA which encodes an immunogen” refers to a polynucleotide,such as a messenger RNA or a replicon (e.g., self-replicating RNA), thatwhen administered to a cell or organism is capable of being translatedinto a polypeptide according to the codon sequence of such RNA.

By “proliferative disease” as used herein is meant any disease,condition, trait, genotype or phenotype characterized by unregulatedcell growth or replication as is known in the art. In one embodiment,the proliferative disease is cancer. In one embodiment, theproliferative disease is a tumor. In one embodiment, the proliferativedisease includes, but are not limited to, e.g., liquid tumors such as,e.g., leukemias, e.g., acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), acute lymphocytic leukemia (ALL), multiplemyeloma, and chronic lymphocytic leukemia; and solid tumors, e.g., AIDSrelated cancers such as Kaposi's sarcoma; breast cancers; bone cancers;brain cancers; cancers of the head and neck, non-Hodgkins lymphoma,adenoma, squamous cell carcinoma, laryngeal carcinoma, gallbladder andbile duct cancers, cancers of the retina, cancers of the esophagus,gastrointestinal cancers, ovarian cancer, uterine cancer, thyroidcancer, testicular cancer, endometrial cancer, melanoma, colorectalcancer, lung cancer, bladder cancer, prostate cancer, lung cancer(including non-small cell lung carcinoma), pancreatic cancer, sarcomas,Wilms' tumor, cervical cancer, head and neck cancer, skin cancers,nasopharyngeal carcinoma, liposarcoma, epithelial carcinoma, renal cellcarcinoma, gallbladder adeno carcinoma, endometrial sarcoma, multidrugresistant cancers. In one embodiment, the proliferative disease includesneovascularization associated with tumor angiogenesis, maculardegeneration (e.g. wet/dry age related macular degeneration), cornealneovascularization, diabetic retinopathy, neovascular glaucoma, myopicdegeneration. In one embodiment, the proliferative disease includesrestenosis and polycystic kidney disease.

By “autoimmune disease” as used herein is meant any disease, condition,trait, genotype or phenotype characterized by autoimmunity as is knownin the art. Autoimmune diseases include, but are not limited to, e.g.,multiple sclerosis, diabetes mellitus, lupus, scleroderms, fibromyalgia,transplantation rejection (e.g. prevention of allograft rejection),pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus,dermatomyositis, myasthenia gravis, lupus erythematosus, multiplesclerosis, and Grave's disease.

By “infectious disease” is meant any disease, disorder or conditionassociated with an infectious agent, such as a virus, bacteria, fungus,prion or parasite. The invention can be used to immunize againstpathogens which cause infectious disease. Examples of such pathogens aregiven below.

By “neurologic disease” is meant any disease, disorder, or conditionaffecting the central or peripheral nervous system. Neurologic diseasesinclude, but are not limited to, diseases or disorders of either theperipheral or the central nervous system including, e.g., Alzheimer'sDisease, Aneurysm, Brain Injury, Carpal Tunnel Syndrome, CerebralAneurysm, Chronic Pain, Creutzfeldt-Jakob Disease, Epilepsy,Huntington's Disease, Meningitis, Seizure Disorders, and otherneurologic diseases, disorders and syndromes.

By “respiratory disease” is meant any disease or condition affecting therespiratory tract. Respiratory diseases include, but are not limited to,e.g., asthma, chronic obstructive pulmonary disease (COPD), allergicrhinitis, sinusitis, allergies, impeded respiration, respiratorydistress syndrome, cystic fibrosis, pulmonary hypertension orvasoconstriction and emphysema.

By “cardiovascular disease” is meant and disease or condition affectingthe heart and vasculature. Cardiovascular diseases include, but are notlimited to, e.g., coronary heart disease (CHD), cerebrovascular disease(CVD), aortic stenosis, peripheral vascular disease, myocardialinfarction (heart attack), arrhythmia, ischemia, and congestive heartfailure.

By “ocular disease” as used herein is meant any disease, condition,trait, genotype or phenotype of the eye and related structures. Oculardiseases include, but are not limited to, e.g., cystoid macular edema,diabetic retinopathy, lattice degeneration, retinal vein occlusion,retinal artery occlusion, macular degeneration (e.g. age related maculardegeneration such as wet AMD or dry AMD), toxoplasmosis, retinitispigmentosa, conjunctival laceration, corneal laceration, glaucoma, andthe like.

By “metabolic disease” is meant any disease or condition affectingmetabolic pathways. Metabolic disease can result in an abnormalmetabolic process, either congenital due to inherited enzyme abnormality(inborn errors of metabolism) or acquired due to disease of an endocrineorgan or failure of a metabolically important organ such as the liver.In one embodiment, metabolic disease includes obesity, insulinresistance, and diabetes (e.g. type I and/or type II diabetes).

By “dermatological disease” is meant any disease or condition of theskin, dermis, or any substructure therein such as a hair, a follicle,etc. Dermatological diseases, disorders, conditions, and traits caninclude psoriasis, ectopic dermatitis, skin cancers such as melanoma andbasal cell carcinoma, hair loss, hair removal and alterations inpigmentation.

By “auditory disease” is meant any disease or condition of the auditorysystem, including the ear, such as the inner ear, middle ear, outer ear,auditory nerve, and any substructures therein. Auditory diseases,disorders, conditions, and traits can include hearing loss, deafness,tinnitus, vertigo, balance and motion disorders.

By “regenerative disease” is meant any disease or condition whereinsufficient cell or tissue generation or regeneration in vivo or invitro prevents the establishment or restoration of proper organ functionbefore or after injury, prevents or slows wound healing or resolution ofulcerative lesions, accelerates ageing, or prevents effective cell-basedtherapy. The term “messenger ribonucleic acid” (messenger RNA, mRNA)refers to a ribonucleic acid (RNA) molecule that mediates the transferof genetic information to ribosomes in the cytoplasm, where it serves asa template for protein synthesis. It is synthesized from a DNA templateduring the process of transcription. See, The American Heritage®Dictionary of the English Language, Fourth Edition (Updated in 2009).Houghton Mifflin Company.

In eukaryotes, mRNA is transcribed in vivo at the chromosomes by thecellular enzyme RNA polymerase. During or after transcription in vivo, a5′ cap (also termed an RNA cap, an RNA 7-methylguanosine cap, or an RNAm7G cap) is added in vivo to the 5′ end of the mRNA. The 5′ cap isterminal 7-methylguanosine residue that is linked through a5′-5′-triphosphate bond to the first transcribed nucleotide. Inaddition, most eukaryotic mRNA molecules have a polyadenylyl moiety(“poly(A) tail”) at the 3′ end of the mRNA molecule. In vivo, theeukaryotic cell adds the poly(A) tail after transcription, often at alength of about 250 adenosine residues (SEQ ID NO: 12). Thus, a typicalmature eukaryotic mRNA has a structure that begins at the 5′ end with anmRNA cap nucleotide followed by a 5′ untranslated region (5′UTR) ofnucleotides, then an open reading frame that begins with a start codonwhich is an AUG triplet of nucleotide bases, that is the coding sequencefor a protein, and that ends with a stop codon that may be a UAA, UAG,or UGA triplet of nucleotide bases, then a 3′ untranslated region(3′UTR) of nucleotides and ending with a poly-adenosine tail. While thefeatures of the typical mature eukaryotic mRNA are made naturally in aeukaryotic cell in vivo, the same or structurally and functionallyequivalent features can be made in vitro using the methods of molecularbiology. Accordingly, any RNA having the structure similar to a typicalmature eukaryotic mRNA can function as a mRNA and is within the scope ofthe term “messenger ribonucleic acid”.

The mRNA molecule is generally of a size that it can be encapsulated ina lipid nanoparticle of the invention. While the size of a mRNA moleculevaries in nature depending upon the identity of the mRNA species thatencodes for a particular protein, an average size for a mRNA molecule isaverage mRNA size is 500-10,000 bases.

DNA can exist in at least two forms, which have different sizes. Thefirst form of DNA is a very large-sized polymer called a chromosome. Achromosome contains the genetic information for many or most of theproteins in a cell and also contains information whereby the cell cancontrol the replication of the DNA molecule. A bacterial cell maycontain one or more chromosome. A eukaryotic cell usually contains morethan one cell chromosome, each chromosome

The second form of DNA is a shorter sized form. Many DNA molecules ofthe second form are of a size that it can be encapsulated in a lipidnanoparticle of the invention. Some of these shorter forms of DNA can beof a size to usefully encode for proteins. Examples of these second,shorter, useful forms of DNA include plasmids and other vectors. For afuller description, see, Alberts B et al. (2007) Molecular Biology ofthe Cell, Fifth Edition, Garland Science.

A plasmid is a small DNA molecule that is physically separate from, andcan replicate independently of, chromosomal DNA within a cell. Plasmidscommonly exist in vivo as small circular, double-stranded DNA molecules.In nature, plasmids carry genes that can be transcribed and translatedto proteins that may benefit survival of an organism (e.g. antibioticresistance). In nature, plasmids can frequently be transmitted from oneorganism to another by horizontal gene transfer. Artificial orrecombinant plasmids are widely used in molecular biology, serving topermit the replication of recombinant DNA sequences and the expressionof useful proteins within host organisms. Plasmid sizes can vary fromabout 1 to over 25 kilobase pairs. A recombinant plasmid can berecombinantly made to be of a size that it can be encapsulated in alipid nanoparticle of the invention.

In molecular biology, a vector is a DNA molecule used as a vehicle toartificially carry genetic material from one cell or from a biochemicalreaction in vitro into another cell, where the DNA can be replicatedand/or expressed. A vector containing foreign DNA is termed recombinant.Among the types of useful vectors are plasmids and viral vectors.Insertion of a vector into the target cell is usually calledtransformation for bacterial cells, transfection for eukaryotic cells,although insertion of a viral vector is often called transduction.

Viral vectors are generally recombinant viruses carrying modified viralDNA or RNA that has been rendered noninfectious, but that still containviral promoters and also the transgene, thus allowing for translation ofthe transgene through a viral promoter. Viral vectors, in someembodiments, are designed for permanent incorporation of the insert intothe host genome (integrate), and thus leave distinct genetic markers inthe host genome after incorporating the transgene. A viral vector can berecombinantly made to be of a size that it can be encapsulated in alipid nanoparticle of the invention.

The term “short interfering nucleic acid” (siNA) as used herein refersto any nucleic acid molecule capable of inhibiting or down regulatinggene expression or viral replication by mediating RNA interference(RNAi) or gene silencing in a sequence-specific manner. It includesshort interfering RNA (siRNA), microRNA (miRNA), short interferingoligonucleotides and chemically-modified short interfering nucleic acidmolecules. siRNAs are responsible for RNA interference, the process ofsequence-specific post-transcriptional gene silencing in animals andplants. siRNAs are generated by ribonuclease III cleavage from longerdouble-stranded RNA (dsRNA) which are homologous to, or specific to, thesilenced gene target.

The term “RNA interference” (RNAi) is a post-transcriptional, targetedgene-silencing technique that uses a RNAi agent to degrade messenger RNA(mRNA) containing a sequence which is the same as or very similar to theRNAi agent. See: Zamore and Haley, 2005, Science, 309, 1519-1524; Zamoreet al., 2000, Cell, 101, 25-33; Elbashir et al., 2001, Nature, 411,494-498; and Kreutzer et al., PCT Publication WO 00/44895; Fire, PCTPublication WO 99/32619; Mello and Fire, PCT Publication WO 01/29058;and the like.

As used herein, RNAi is equivalent to other terms used to describesequence specific RNA interference, such as post transcriptional genesilencing, translational inhibition, transcriptional inhibition, orepigenetics. For example, the formulations containing lipids of theinvention can be used in conjunction with siNA molecules toepigenetically silence genes at both the post-transcriptional leveland/or the pre-transcriptional level. In a non-limiting example,modulation of gene expression by siNA molecules can result from siNAmediated cleavage of RNA (either coding or non-coding RNA) via RISC, oralternately, translational inhibition as is known in the art. In anotherembodiment, modulation of gene expression by siNA can result fromtranscriptional inhibition such as is reported e.g., in Janowski et al.,2005, Nature Chemical Biology, 1, 216-222.

The term “RNAi inhibitor” is any molecule that can down modulate (e.g.reduce or inhibit) RNA interference function or activity in a cell orpatient. An RNAi inhibitor can down regulate, reduce or inhibit RNAi(e.g. RNAi mediated cleavage of a target polynucleotide, translationalinhibition, or transcriptional silencing) by interaction with orinterfering with the function of any component of the RNAi pathway,including protein components such as RISC, or nucleic acid componentssuch as miRNAs or siRNAs. An RNAi inhibitor can be a siNA molecule, anantisense molecule, an aptamer, or a small molecule that interacts withor interferes with the function of RISC, a miRNA, or a siRNA or anyother component of the RNAi pathway in a cell or patient. By inhibitingRNAi (e.g. RNAi mediated cleavage of a target polynucleotide,translational inhibition, or transcriptional silencing), an RNAiinhibitor can be used to modulate (e.g, up-regulate or down-regulate)the expression of a target gene. In one embodiment, an RNA inhibitor isused to up-regulate gene expression by interfering with (e.g. reducingor preventing) endogenous down-regulation or inhibition of geneexpression through translational inhibition, transcriptional silencing,or RISC mediated cleavage of a polynucleotide (e.g. mRNA). Byinterfering with mechanisms of endogenous repression, silencing, orinhibition of gene expression, RNAi inhibitors of the invention cantherefore be used to up-regulate gene expression for the treatment ofdiseases or conditions resulting from a loss of function. The term “RNAiinhibitor” is used interchangeably with the term “siNA” in variousembodiments herein.

The term “enzymatic nucleic acid” as used herein refers to a nucleicacid molecule that has complementarity in a substrate binding region toa specified gene target, and also has an enzymatic activity that acts tospecifically cleave a target RNA, thereby inactivating the target RNAmolecule. The complementary regions allow sufficient hybridization ofthe enzymatic nucleic acid molecule to the target RNA and thus permitcleavage. Complementarity of 100% is preferred, but complementarity aslow as 50-75% can also be useful in this invention (see e.g., Werner andUhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al.,1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). The nucleic acidscan be modified at the base, sugar, and/or phosphate groups. The termenzymatic nucleic acid is used interchangeably with phrases such asribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme oraptamer-binding ribozyme, regulatable ribozyme, catalyticoligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease,endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of theseterminologies describe nucleic acid molecules with enzymatic activity.The key features of an enzymatic nucleic acid molecule are that it has aspecific substrate binding site that is complementary to one or more ofthe target nucleic acid regions, and that it has nucleotide sequenceswithin or surrounding that substrate binding site that impart a nucleicacid cleaving and/or ligation activity to the molecule (see, e.g., Cechet al., U.S. Pat. No. 4,987,071; Cech et al., 1988, 260 JAMA 3030).Ribozymes and enzymatic nucleic acid molecules of the invention can bechemically modified, e.g., as described in the art and elsewhere herein.

The term “antisense nucleic acid”, as used herein, refers to anon-enzymatic nucleic acid molecule that binds to target RNA by means ofRNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993Nature 365, 566) interactions and alters the activity of the target RNA(for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf etal., U.S. Pat. No. 5,849,902). Antisense DNA can be synthesizedchemically or expressed via the use of a single stranded DNA expressionvector or equivalent thereof. Antisense molecules of the invention canbe chemically modified, e.g. as described in the art.

The term “RNase H activating region” as used herein, refers to a region(generally greater than or equal to 4-25 nucleotides in length,preferably from 5-11 nucleotides in length) of a nucleic acid moleculecapable of binding to a target RNA to form a non-covalent complex thatis recognized by cellular RNase H enzyme (see e.g., Arrow et al., U.S.Pat. No. 5,849,902; Arrow et al., U.S. Pat. No. 5,989,912). The RNase Henzyme binds to the nucleic acid molecule-target RNA complex and cleavesthe target RNA sequence.

The term “2-5A antisense chimera” as used herein, refers to an antisenseoligonucleotide containing a 5′-phosphorylated 2′-5′-linked adenylateresidue. These chimeras bind to target RNA in a sequence-specific mannerand activate a cellular 2-5A-dependent ribonuclease that, in turn,cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA90, 1300; Silverman et al., 2000, Methods Enzymol., 313, 522-533; Playerand Torrence, 1998, Pharmacol. Ther., 78, 55-113). 2-5A antisensechimera molecules can be chemically modified, e.g. as described in theart.

The term “triplex forming oligonucleotides” as used herein, refers to anoligonucleotide that can bind to a double-stranded DNA in asequence-specific manner to form a triple-strand helix. Formation ofsuch triple helix structure has been shown to inhibit transcription ofthe targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci.USA 89, 504; Fox, 2000, Curr. Med. Chem., 7, 17-37; Praseuth et. al.,2000, Biochim. Biophys. Acta, 1489, 181-206). Triplex formingoligonucleotide molecules of the invention can be chemically modified,e.g. as described in the art.

The term “decoy RNA” as used herein, refers to an RNA molecule oraptamer that is designed to preferentially bind to a predeterminedligand. Such binding can result in the inhibition or activation of atarget molecule. The decoy RNA or aptamer can compete with a naturallyoccurring binding target for the binding of a specific ligand.Similarly, a decoy RNA can be designed to bind to a receptor and blockthe binding of an effector molecule, or can be designed to bind toreceptor of interest and prevent interaction with the receptor. Decoymolecules of the invention can be chemically modified, e.g. as describedin the art.

The term “single stranded DNA” (ssDNA) as used herein refers to anaturally occurring or synthetic deoxyribonucleic acid moleculecomprising a linear single strand, e.g., a ssDNA can be a sense orantisense gene sequence or EST (Expressed Sequence Tag).

The term “allozyme” as used herein refers to an allosteric enzymaticnucleic acid molecule, including e.g., U.S. Pat. Nos. 5,834,186;5,741,679; 5,589,332; 5,871,914; and PCT publication Nos. WO 00/24931,WO 00/26226, WO 98/27104, and WO 99/29842.

The term “aptamer” as used herein is meant a polynucleotide compositionthat binds specifically to a target molecule, wherein the polynucleotidehas a sequence that differs from a sequence normally recognized by thetarget molecule in a cell. Alternately, an aptamer can be a nucleic acidmolecule that binds to a target molecule where the target molecule doesnot naturally bind to a nucleic acid. The target molecule can be anymolecule of interest. Aptamer molecules of the invention can bechemically modified, e.g. as described in the art.

Formulation of Lipid Compositions

For pharmaceutical use, the lipid compositions of the invention may beadministered by enteral or parenteral routes, including intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), oral,intranasal, rectal, vaginal, buccal, nasopharangeal, gastrointestinal orsublingual administration. The administration may be systemic (e.g., IV)or local (e.g., IM, SC, TD, intranasal, or topical). Topicaladministration may involve, e.g., catheterization, implantation, osmoticpumping, direct injection, dermal/transdermal application, stenting,ear/eye drops or portal vein administration. The compounds of formula(I) should be assessed for their biopharmaceutical properties, such assolubility and solution stability (across pH), permeability, etc., inorder to select the most appropriate dosage form and route ofadministration for treatment of the proposed indication.

The compositions of the invention will generally, but not necessarily,be administered as a formulation in association with one or morepharmaceutically acceptable excipients. The term “excipient” includesany ingredient other than the compound(s) of the invention, the otherlipid component(s) and the biologically active agent. An excipient mayimpart either a functional (e.g drug release rate controlling) and/or anon-functional (e.g. processing aid or diluent) characteristic to theformulations. The choice of excipient will to a large extent depend onfactors such as the particular mode of administration, the effect of theexcipient on solubility and stability, and the nature of the dosageform.

Typical pharmaceutically acceptable excipients include:

-   -   diluents, e.g. lactose, dextrose, sucrose, mannitol, sorbitol,        cellulose and/or glycine;    -   lubricants, e.g. silica, talcum, stearic acid, its magnesium or        calcium salt and/or polyethyleneglycol;    -   binders, e.g. magnesium aluminum silicate, starch paste,        gelatin, tragacanth, methylcellulose, sodium        carboxymethylcellulose and/or polyvinyl pyrrolidone;    -   disintegrants, e.g. starches, agar, alginic acid or its sodium        salt, or effervescent mixtures; and/or    -   absorbants, colorants, flavors and/or sweeteners.

The excipient may be an aqueous solution carrier which may optionallycontain a buffer (e.g. a PBS buffer) and/or a sugar.

A thorough discussion of pharmaceutically acceptable excipients isavailable in Gennaro, Remington: The Science and Practice of Pharmacy2000, 20th edition (ISBN: 0683306472).

The compositions of the invention may be administered orally. Oraladministration may involve swallowing, so that the compound enters thegastrointestinal tract, and/or buccal, lingual, or sublingualadministration by which the compound enters the blood stream directlyfrom the mouth.

The compositions of the invention can be administered parenterally. Thecompounds and compositions of the invention may be administered directlyinto the blood stream, into subcutaneous tissue, into muscle, or into aninternal organ. Suitable means for administration include intravenous,intraarterial, intrathecal, intraventricular, intraurethral,intrasternal, intracranial, intramuscular, intrasynovial andsubcutaneous. Suitable devices for administration include needle(including microneedle) injectors, needle-free injectors and infusiontechniques.

Parenteral formulations are typically aqueous or oily solutions. Wherethe solution is aqueous, excipients such as sugars (including but notrestricted to glucose, mannitol, sorbitol, etc.) salts, carbohydratesand buffering agents (preferably to a pH of from 3 to 9), but, for someapplications, they may be more suitably formulated as a sterilenon-aqueous solution or as a dried form to be used in conjunction with asuitable vehicle such as sterile, pyrogen-free water (WFI).

Parenteral formulations may include implants derived from degradablepolymers such as polyesters (i.e. polylactic acid, polylactide,polylactide-co-glycolide, polycapro-lactone, polyhydroxybutyrate),polyorthoesters and polyanhydrides. These formulations may beadministered via surgical incision into the subcutaneous tissue,muscular tissue or directly into specific organs.

The preparation of parenteral formulations under sterile conditions,e.g., by lyophilisation, may readily be accomplished using standardpharmaceutical techniques well known to the skilled person.

The solubility of the compounds and compositions used in the preparationof parenteral solutions may be increased by the use of appropriateformulation techniques, such as the incorporation of co-solvents and/orsolubility-enhancing agents such as surfactants, micelle structures andcyclodextrins.

The compositions of the invention can be administered intranasally or byinhalation, typically in the form of a dry powder (either alone, as amixture, e.g., in a dry blend with lactose, or as a mixed componentparticle, e.g., mixed with phospholipids, such as phosphatidylcholine)from a dry powder inhaler, as an aerosol spray from a pressurisedcontainer, pump, spray, atomiser (preferably an atomiser usingelectrohydrodynamics to produce a fine mist), or nebuliser, with orwithout the use of a suitable propellant, such as1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane, or asnasal drops. For intranasal use, the powder may comprise a bioadhesiveagent, e.g., chitosan or cyclodextrin.

The pressurized container, pump, spray, atomizer, or nebuliser containsa solution or suspension of the compound(s) of the invention comprising,e.g., ethanol, aqueous ethanol, or a suitable alternative agent fordispersing, solubilising, or extending release of the compositions ofthe invention, a propellant(s) as solvent and an optional surfactant,such as sorbitan trioleate, oleic acid, or an oligolactic acid.

Prior to use in a dry powder or suspension formulation, the compositionis micronised to a size suitable for delivery by inhalation (typicallyless than 5 microns). This may be achieved by any appropriatecomminuting method, such as spiral jet milling, fluid bed jet milling,supercritical fluid processing to form nanoparticles, high pressurehomogenisation, or spray drying.

Capsules (made, e.g., from gelatin or hydroxypropylmethylcellulose),blisters and cartridges for use in an inhaler or insufflator may beformulated to contain a powder mix of the compound or composition of theinvention, a suitable powder base such as lactose or starch and aperformance modifier such as I-leucine, mannitol, or magnesium stearate.The lactose may be anhydrous or in the form of the monohydrate,preferably the latter. Other suitable excipients include dextran,glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.

Formulations for inhaled/intranasal administration may be formulated tobe immediate and/or modified release using, e.g., PGLA. Modified releaseformulations include delayed-, sustained-, pulsed-, controlled-,targeted and programmed release.

Suitable formulations for transdermal application include atherapeutically effective amount of a compound or composition of theinvention with carrier. Advantageous carriers include absorbablepharmacologically acceptable solvents to assist passage through the skinof the host. Characteristically, transdermal devices are in the form ofa bandage comprising a backing member, a reservoir containing thecompound optionally with carriers, optionally a rate controlling barrierto deliver the compound to the skin of the host at a controlled andpredetermined rate over a prolonged period of time, and means to securethe device to the skin.

Lipid compositions of the invention are administered in any of a numberof ways, including parenteral, intravenous, systemic, local, oral,intratumoral, intramuscular, subcutaneous, intraperitoneal, inhalation,or any such method of delivery. In one embodiment, the compositions areadministered parenterally, i.e., intraarticularly, intravenously,intraperitoneally, subcutaneously, or intramuscularly. In a specificembodiment, the liposomal compositions are administered by intravenousinfusion or intraperitoneally by a bolus injection.

Lipid compositions of the invention can be formulated as pharmaceuticalcompositions suitable for delivery to a subject. The pharmaceuticalcompositions of the invention will often further comprise one or morebuffers (e.g., neutral buffered saline or phosphate buffered saline),carbohydrates (e.g., glucose, mannose, sucrose, dextrose or dextrans),mannitol, proteins, polypeptides or amino acids such as glycine,antioxidants, bacteriostats, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide), solutes that renderthe formulation isotonic, hypotonic or weakly hypertonic with the bloodof a recipient, suspending agents, thickening agents and/orpreservatives. Alternatively, compositions of the present invention maybe formulated as a lyophilizate.

Suitable formulations for use in the present invention can be found,e.g., in Remington's Pharmaceutical Sciences, Mack Publishing Company,Philadelphia, Pa., 17.sup.th Ed. (1985). Often, compositions willcomprise a solution of the lipid nanoparticles suspended in anacceptable carrier, such as an aqueous carrier.

In one embodiment, this invention provides for a pharmaceuticalcomposition (i.e. formulation) comprising a lipid composition of theinvention and a pharmaceutically acceptable carrier or excipient. Inanother embodiment at least one other lipid component is present in thelipid composition. In another embodiment the lipid composition is in theform of a liposome. In another embodiment the lipid composition is inthe form of a lipid nanoparticle. In another embodiment the lipidcomposition is suitable for delivery to the liver. In another embodimentthe lipid composition is suitable for delivery to a tumor. In anotherembodiment the lipid composition is suitable for local deliveryapplications (eye, ear, skin, lung); delivery to muscle (i.m.), fat, orsub cutaneous cells (s.c. dosing). In another embodiment thebiologically active agent is a RNA or DNA.

For immunization purposes a composition will generally be prepared as aninjectable, and will be administered by injection (e.g. by intramuscularinjection).

The invention also provides a delivery device (e.g. syringe, nebuliser,sprayer, inhaler, dermal patch, etc.) containing a composition of theinvention. This device can be used to administer a pharmaceuticalcomposition to a subject e.g. to a human for immunization.

Cells and Organs Targeted by the Invention

The compounds, compositions, methods and uses of the invention can beused to deliver a biologically active agent to one or more of thefollowing in a patient:

the liver or liver cells (e.g. hepatocytes);

a kidney or kidney cells;

a tumor or tumor cells;

the CNS or CNS cells (Central Nervous System, e.g. brain and/or spinalcord);

the PNS or PNS cells (Peripheral Nervous System);

a lung or lung cells;

the vasculature or vascular cells;

the skin or skin cells (e.g. dermis cells and/or follicular cells);

an eye or ocular cells (e.g. macula, fovea, cornea, retina), and

an ear or cells of the ear (e.g. cells of the inner ear, middle earand/or outer ear).

The compounds, compositions, methods and uses of the invention can alsobe used to deliver a biologically active agent (e.g. RNA which encodesan immunogen) to cells of the immune system.

In one embodiment, the compounds, compositions, methods and uses of theinvention are for delivering a biologically active agent to liver cells(e.g. hepatocytes). In one embodiment, the compounds, compositions,methods and uses of the invention are for delivering a biologicallyactive agent to a tumor or to tumor cells (e.g. a primary tumor ormetastatic cancer cells). In another embodiment, the compounds,compositions, methods and uses are for delivering a biologically activeagent to the skin adipose, muscle and lymph nodes (i.e. sc dosing).

For delivery of a biologically active agent to the liver or liver cells,in one embodiment a composition of the invention is contacted with theliver or liver cells of the patient as is generally known in the art,such as via parental administration (e.g. intravenous, intramuscular,subcutaneous administration) or local administration (e.g. directinjection, portal vein injection, catheterization, stenting), tofacilitate delivery.

For delivery of a biologically active agent to the kidney or kidneycells, in one embodiment a composition of the invention is contactedwith the kidney or kidney cells of the patient as is generally known inthe art, such as via parental administration (e.g. intravenous,intramuscular, subcutaneous administration) or local administration(e.g. direct injection, catheterization, stenting), to facilitatedelivery.

For delivery of a biologically active agent to a tumor or tumor cells,in one embodiment a composition of the invention is contacted with thetumor or tumor cells of the patient as is generally known in the art,such as via parental administration (e.g. intravenous, intramuscular,subcutaneous administration) or local administration (e.g. directinjection, catheterization, stenting), to facilitate delivery.

For delivery of a biologically active agent to the CNS or CNS cells(e.g. brain cells and/or spinal cord cells), in one embodiment acomposition of the invention is contacted with the CNS or CNS cells(e.g. brain cells and/or spinal cord cells) of the patient as isgenerally known in the art, such as via parental administration (e.g.intravenous, intramuscular, subcutaneous administration) or localadministration (e.g. direct injection, catheterization, stenting,osmotic pump administration (e.g. intrathecal or ventricular)), tofacilitate delivery.

For delivery of a biologically active agent to the PNS or PNS cells, inone embodiment a composition of the invention is contacted with the PNSor PNS cells of the patient as is generally known in the art, such asvia parental administration (e.g. intravenous, intramuscular,subcutaneous administration) or local administration (e.g. directinjection), to facilitate delivery.

For delivery of a biologically active agent to a lung or lung cells, inone embodiment a composition of the invention is contacted with the lungor lung cells of the patient as is generally known in the art, such asvia parental administration (e.g. intravenous, intramuscular,subcutaneous administration) or local administration (e.g. pulmonaryadministration directly to lung tissues and cells), to facilitatedelivery.

For delivery of a biologically active agent to the vasculature orvascular cells, in one embodiment a composition of the invention iscontacted with the vasculature or vascular cells of the patient as isgenerally known in the art, such as via parental administration (e.g.intravenous, intramuscular, subcutaneous administration) or localadministration (e.g. clamping, catheterization, stenting), to facilitatedelivery.

For delivery of a biologically active agent to the skin or skin cells(e.g. dermis cells and/or follicular cells), in one embodiment acomposition of the invention is contacted with the skin or skin cells(e.g. dermis cells and/or follicular cells) of the patient as isgenerally known in the art, such as via parental administration (e.g.intravenous, intramuscular, subcutaneous administration) or localadministration (e.g. direct dermal application, iontophoresis), tofacilitate delivery.

For delivery of a biologically active agent to an eye or ocular cells(e.g. macula, fovea, cornea, retina), in one embodiment a composition ofthe invention is contacted with the eye or ocular cells (e.g. macula,fovea, cornea, retina) of the patient as is generally known in the art,such as via parental administration (e.g. intravenous, intramuscular,subcutaneous administration) or local administration (e.g. directinjection, intraocular injection, periocular injection, subretinal,iontophoresis, use of eyedrops, implants), to facilitate delivery.

For delivery of a biologically active agent to an ear or cells of theear (e.g. cells of the inner ear, middle ear and/or outer ear), in oneembodiment composition of the invention is contacted with the ear orcells of the ear (e.g. cells of the inner ear, middle ear and/or outerear) of the patient as is generally known in the art, such as viaparental administration (e.g. intravenous, intramuscular, subcutaneousadministration) or local administration (e.g. direct injection), tofacilitate delivery.

For delivery of a biologically active agent (e.g. RNA encoding animmunogen) to cells of the immune system (e.g. antigen-presenting cells,including professional antigen presenting cells), in one embodimentcomposition of the invention is delivered intramuscularly, after whichimmune cells can infiltrate the delivery site and process delivered RNA.Such immune cells can include macrophages (e.g. bone marrow derivedmacrophages), dendritic cells (e.g. bone marrow derived plasmacytoiddendritic cells and/or bone marrow derived myeloid dendritic cells),monocytes (e.g. human peripheral blood monocytes), etc. (e.g. seeWO2012/006372).

Immunization According to the Invention

For immunization purposes, in some embodiments, the inventionencompasses delivering a RNA that encodes an immunogen. The immunogenelicits an immune response which recognizes the immunogen, and so can beused to provide immunity against a pathogen, or against an allergen, oragainst a tumor antigen. Immunising against disease and/or infectioncaused by a pathogen is preferred.

The RNA is delivered with a lipid composition of the invention (e.g.formulated as a liposome or LNP). In some embodiments, the inventionutilises liposomes within which immunogen-encoding RNA is encapsulated.Encapsulation within liposomes can protect RNA from RNase digestion. Theencapsulation efficiency does not have to be 100%. Presence of externalRNA molecules (e.g. on the exterior surface of liposome) or “naked” RNAmolecules (RNA molecules not associated with a liposome) is acceptable.Preferably, for a composition comprising liposomes and RNA molecules, atleast half of the RNA molecules (e.g., at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% of the RNA molecules) areencapsulated in liposomes.

RNA molecules may also be complexed with LNPs. For example, it is notnecessary that the lipid forms liposomes (with aqueous core) only. Somelipid nanoparticles may comprise a lipid core (e.g., the composition maycomprise a mixture of liposomes and nanoparticles with a lipid core). Insuch cases, the RNA molecules may be encapsulated by LNPs that have anaqueous core, and complexed with the LNPs that have a lipid core bynon-covalent interactions (e.g., ionic interactions between negativelycharged RNA and cationic lipid). Encapsulation and complexation withLNPs (whether with a lipid or aqueous core) can protect RNA from RNasedigestion. The encapsulation/complexation efficiency does not have to be100%. Presence of “naked” RNA molecules (RNA molecules not associatedwith a liposome) is acceptable. Preferably, for a composition comprisinga population of LNPs and a population of RNA molecules, at least half ofthe population of RNA molecules (e.g., at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% of the RNA molecules) areeither encapsulated in LNPs, or complexed with LNPs.

Liposomes and LNPs

Liposomes are usually divided into three groups: multilamellar vesicles(MLV); small unilamellar vesicles (SUV); and large unilamellar vesicles(LUV). MLVs have multiple bilayers in each vesicle, forming severalseparate aqueous compartments. SUVs and LUVs have a single bilayerencapsulating an aqueous core; SUVs typically have a diameter <50 nm,and LUVs have a diameter >50 nm. For delivery of immunogen-coding RNA,preferred range of diameters is in the range of 60-180 nm, and morepreferably in the range of 80-160 nm.

The lipid composition can also be LNPs. The composition can comprise amixture of nanoparticles having an aqueous core and nanoparticles havinga lipid core. For delivery of immunogen-coding RNA, preferred range ofdiameters is in the range of 60-180 nm, and in more particularembodiments, in the range of 80-160 nm.

A liposome or LNP can be part of a composition comprising a populationof liposomes or LNPs, and the liposomes or LNPs within the populationcan have a range of diameters. For a composition comprising a populationof liposomes or LNPs with different diameters, it is preferred that (i)at least 80% by number of the liposomes or LNPs have diameters in therange of 60-180 nm, e.g., in the range of 80-160 nm, (ii) the averagediameter (by intensity e.g. Z-average) of the population is ideally inthe range of 60-180 nm, e.g., in the range of 80-160 nm; and/or (iii)the diameters within the plurality have a polydispersity index <0.2.

To obtain liposomes or LNPs with the desired diameter(s), mixing can beperformed using a process in which two feed streams of aqueous RNAsolution are combined in a single mixing zone with one stream of anethanolic lipid solution, all at the same flow rate e.g. in amicrofluidic channel.

Useful mixtures of lipids, for forming lipid compositions (e.g.,liposomes or LNPs) for immunization uses, comprise: a lipid of formula(I); cholesterol; and a PEGylated lipid, such as PEG-DMG i.e.PEG-conjugated1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol). This mixture may also include a neutral zwitterionic lipid,such as DSPC (1,2-Diastearoyl-sn-glycero-3-phosphocholine) or DPyPE.These (and other) mixtures are used in the examples.

In certain embodiments, the lipid compositions provided by the invention(such as liposomes or LNPs) have adjuvant activity, i.e., in the absenceof an immunogen, such as protein antigen or a nucleic acid (DNA or RNA),such as a nucleic acid encoding such an antigen. Therefore the lipidsand lipid composition provided by the invention can be formulated withany manner of antigen, e.g., polypeptide, nucleic acid, small molecule,et cetera. Thus, in some embodiments, compositions provided by theinvention can be used in methods of generating an immune response to animmunogen, e.g., by administering a composition comprising a lipid orlipid composition provided by the invention together with an immunogen.

RNA Molecules

After in vivo administration of an immunization composition, thedelivered RNA is released and is translated inside a cell to provide theimmunogen in situ. In certain embodiments, the the RNA is plus (“+”)stranded, so it can be translated by cells without needing anyintervening replication steps such as reverse transcription. It may alsobind to TLR7 receptors expressed by immune cells, thereby initiating anadjuvant effect. Additionally or alternatively, the RNA may bind otherreceptors such as RIG I, MDA5, or RIG I and MDA5.

In certain embodiments, the RNA is a self-replicating RNA. Aself-replicating RNA molecule (replicon) can, when delivered to avertebrate cell even without any proteins, lead to the production ofmultiple daughter RNAs by transcription from itself (via an antisensecopy which it generates from itself). A self-replicating RNA molecule isthus, in certain embodiments, a (+) strand molecule which can bedirectly translated after delivery to a cell, and this translationprovides a RNA-dependent RNA polymerase which then produces bothantisense and sense transcripts from the delivered RNA. Thus thedelivered RNA leads to the production of multiple daughter RNAs. Thesedaughter RNAs, as well as collinear subgenomic transcripts, may betranslated themselves to provide in situ expression of an encodedimmunogen, or may be transcribed to provide further transcripts with thesame sense as the delivered RNA which are translated to provide in situexpression of the immunogen. The overall result of this sequence oftranscriptions is a huge amplification in the number of the introducedreplicon RNAs and so the encoded immunogen becomes a major polypeptideproduct of the host cells.

One suitable system for achieving self-replication is to use analphavirus-based RNA replicon. These (+) stranded replicons aretranslated after delivery to a cell to give of a replicase (orreplicase-transcriptase). The replicase is translated as a polyproteinwhich auto cleaves to provide a replication complex which createsgenomic (−) strand copies of the (+) strand delivered RNA. These (−)strand transcripts can themselves be transcribed to give further copiesof the +stranded parent RNA and also to give a subgenomic transcriptwhich encodes the immunogen. Translation of the subgenomic transcriptthus leads to in situ expression of the immunogen by the infected cell.Suitable alphavirus replicons can use a replicase from a sindbis virus,a semliki forest virus, an eastern equine encephalitis virus, aVenezuelan equine encephalitis virus, etc. Mutant or wild-type virusessequences can be used e.g. the attenuated TC83 mutant of VEEV has beenused in replicons.

A preferred self-replicating RNA molecule thus encodes (i) aRNA-dependent RNA polymerase which can transcribe RNA from theself-replicating RNA molecule and (ii) an immunogen. The polymerase canbe an alphavirus replicase e.g. comprising one or more of alphavirusproteins nsP1, nsP2, nsP3 and nsP4.

Whereas natural alphavirus genomes encode structural virion proteins inaddition to the non structural replicase polyprotein, in particularembodiments, a self-replicating RNA molecule of the invention does notencode alphavirus structural proteins. Thus a particular selfreplicating RNA can lead to the production of genomic RNA copies ofitself in a cell, but not to the production of RNA-containing virions.The inability to produce these virions means that, unlike a wild-typealphavirus, the self-replicating RNA molecule cannot perpetuate itselfin infectious form. The alphavirus structural proteins which arenecessary for perpetuation in wild-type viruses are absent from selfreplicating RNAs of the invention and their place is taken by gene(s)encoding the immunogen of interest, such that the subgenomic transcriptencodes the immunogen rather than the structural alphavirus virionproteins.

Thus a self-replicating RNA molecule useful with the invention may havetwo open reading frames. One open reading frame encodes a replicase,e.g., the first, (5′) open reading frame; the other open reading frameencodes an immunogen, e.g., the second, (3′) open reading frame. In someembodiments the RNA may have additional (e.g. downstream) open readingframes e.g. to encode further immunogens (see below) or to encodeaccessory polypeptides.

A self-replicating RNA molecule can have a 5′ sequence which iscompatible with the encoded replicase.

Self-replicating RNA molecules can have various lengths but they aretypically 5000-25000 nucleotides long e.g. 8000-15000 nucleotides, or9000-12000 nucleotides. Thus the RNA is longer than seen in siRNA orconventional mRNA delivery. In some embodiments, the self-replicatingRNA is greater than about 2000 nucleotides, such as greater than about:9000, 12000, 15000, 18000, 21000, 24000, or more nucleotides long

A RNA molecule may have a 5′ cap (e.g. a 7-methylguanosine). This capcan enhance in vivo translation of the RNA.

The 5′ nucleotide of a RNA molecule useful with the invention may have a5′ triphosphate group. In a capped RNA this may be linked to a7-methylguanosine via a 5′-to-5′ bridge. A 5′ triphosphate can enhanceRIG-I binding and thus promote adjuvant effects.

A RNA molecule may have a 3′ poly A tail. It may also include a poly Apolymerase recognition sequence (e.g. AAUAAA) near its 3′ end.

A RNA molecule useful with the invention for immunization purposes willtypically be single-stranded. Single-stranded RNAs can generallyinitiate an adjuvant effect by binding to TLR7, TLR8, RNA helicasesand/or PKR. RNA delivered in double-stranded form (dsRNA) can bind toTLR3, and this receptor can also be triggered by dsRNA which is formedeither during replication of a single-stranded RNA or within thesecondary structure of a single-stranded RNA.

RNA molecules for immunization purposes can conveniently be prepared byin vitro transcription (IVT). IVT can use a (cDNA) template created andpropagated in plasmid form in bacteria, or created synthetically (forexample by gene synthesis and/or polymerase chain-reaction (PCR)engineering methods). For instance, a DNA-dependent RNA polymerase (suchas the bacteriophage T7, T3 or SP6 RNA polymerases) can be used totranscribe the RNA from a DNA template. Appropriate capping and poly Aaddition reactions can be used as required (although the replicon'spoly-A is usually encoded within the DNA template). These RNApolymerases can have stringent requirements for the transcribed 5′nucleotide(s) and in some embodiments these requirements must be matchedwith the requirements of the encoded replicase, to ensure that the IVTtranscribed RNA can function efficiently as a substrate for itsself-encoded replicase.

As discussed in WO2011/005799, the self-replicating RNA can include (inaddition to any 5′ cap structure) one or more nucleotides having amodified nucleobase. For instance, a self-replicating RNA can includeone or more modified pyrimidine nucleobases, such as pseudouridineand/or 5 methylcytosine residues. In some embodiments, however, the RNAincludes no modified nucleobases, and may include no modifiednucleotides i.e. all of the nucleotides in the RNA are standard A, C, Gand U ribonucleotides (except for any 5′ cap structure, which mayinclude a 7′ methylguanosine). In other embodiments, the RNA may includea 5′ cap comprising a 7′ methylguanosine, and the first 1, 2 or 3 5′ribonucleotides may be methylated at the 2′ position of the ribose.

A RNA used with the invention for immunization purposes ideally includesonly phosphodiester linkages between nucleosides, but in someembodiments it can contain phosphoramidate, phosphorothioate, and/ormethylphosphonate linkages.

The invention includes embodiments where multiple species of RNAs areformulated with a lipid composition provided by the invention, such as2, 3, 4, 5, 6, 7, 8, 9, 10, or more species of RNA, including differentclasses of RNA (such as mRNA, siRNA, self-replicating RNAs, andcombinations thereof).

Immunogens

RNA molecules used with the invention for immunization purposes, in someembodiments, encode a polypeptide immunogen. In these embodiments, afteradministration, the RNA is translated in vivo and the immunogen canelicit an immune response in the recipient. The immunogen may elicit animmune response against a pathogen (e.g. a bacterium, a virus, a fungusor a parasite) but, in some embodiments, it elicits an immune responseagainst an allergen or a tumor antigen. The immune response may comprisean antibody response (usually including IgG) and/or a cell mediatedimmune response. The polypeptide immunogen will typically elicit animmune response which recognises the corresponding pathogen (or allergenor tumor) polypeptide, but in some embodiments the polypeptide may actas a mimotope to elicit an immune response which recognises asaccharide. The immunogen will typically be a surface polypeptide e.g.an adhesin, a hemagglutinin, an envelope glycoprotein, a spikeglycoprotein, etc.

The RNA molecule can encode a single polypeptide immunogen or multiplepolypeptides. Multiple immunogens can be presented as a singlepolypeptide immunogen (fusion polypeptide) or as separate polypeptides.If immunogens are expressed as separate polypeptides from a repliconthen one or more of these may be provided with an upstream IRES or anadditional viral promoter element. Alternatively, multiple immunogensmay be expressed from a polyprotein that encodes individual immunogensfused to a short autocatalytic protease (e.g. foot-and-mouth diseasevirus 2A protein), or as inteins.

In certain embodiments, polypeptide immunogens (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more immunogens) may be used, either alone or togetherwith a RNA molecule, such as a self-replicating RNA, encoding one ormore immunogens (either the same or different as the polypeptideimmunogens).

In some embodiments the immunogen elicits an immune response against oneof these bacteria:

Neisseria meningitidis: useful immunogens include, but are not limitedto, membrane proteins such as adhesins, autotransporters, toxins, ironacquisition proteins, and factor H binding protein. A combination ofthree useful polypeptides is disclosed in Giuliani et al. (2006) ProcNatl Acad Sci USA 103(29):10834-9.

Streptococcus pneumoniae: useful polypeptide immunogens are disclosed inWO2009/016515. These include, but are not limited to, the RrgB pilussubunit, the beta-N-acetyl-hexosaminidase precursor (spr0057), spr0096,General stress protein GSP-781 (spr2021, SP2216), serine/threoninekinase StkP (SP1732), and pneumococcal surface adhesin PsaA.

Streptococcus pyogenes: useful immunogens include, but are not limitedto, the polypeptides disclosed in WO02/34771 and WO2005/032582.

Moraxella catarrhalis.

Bordetella pertussis: Useful pertussis immunogens include, but are notlimited to, pertussis toxin or toxoid (PT), filamentous haemagglutinin(FHA), pertactin, and agglutinogens 2 and 3.

Staphylococcus aureus: Useful immunogens include, but are not limitedto, the polypeptides disclosed in WO2010/119343, such as a hemolysin,esxA, esxB, ferrichrome-binding protein (sta006) and/or the sta011lipoprotein.

Clostridium tetani: the typical immunogen is tetanus toxoid.

Cornynebacterium diphtheriae: the typical immunogen is diphtheriatoxoid.

Haemophilus influenzae: Useful immunogens include, but are not limitedto, the polypeptides disclosed in WO2006/110413 and WO2005/111066.

Pseudomonas aeruginosa

Streptococcus agalactiae: useful immunogens include, but are not limitedto, the polypeptides disclosed in WO02/34771.

Chlamydia trachomatis: Useful immunogens include, but are not limitedto, PepA, LcrE, ArtJ, DnaK, CT398, OmpH-like, L7/L12, OmcA, AtoS, CT547,Eno, HtrA and MurG (e.g. as disclosed in WO2005/002619). LcrE(WO2006/138004) and HtrA (WO2009/109860) are two preferred immunogens.

Chlamydia pneumoniae: Useful immunogens include, but are not limited to,the polypeptides disclosed in WO02/02606.

Helicobacter pylori: Useful immunogens include, but are not limited to,CagA, VacA, NAP, and/or urease (WO03/018054).

Escherichia coli: Useful immunogens include, but are not limited to,immunogens derived from enterotoxigenic E. coli (ETEC),enteroaggregative E. coli (EAggEC), diffusely adhering E. coli (DAEC),enteropathogenic E coli (EPEC), extraintestinal pathogenic E. coli(ExPEC) and/or enterohemorrhagic E. coli (EHEC). ExPEC strains includeuropathogenic E. coli (UPEC) and meningitis/sepsis-associated E. coli(MNEC). Useful UPEC immunogens are disclosed in WO2006/091517 andWO2008/020330. Useful MNEC immunogens are disclosed in WO2006/089264. Auseful immunogen for several E. coli types is AcfD (WO2009/104092).

Bacillus anthracis

Yersinia pestis: Useful immunogens include, but are not limited to,those disclosed in WO2007/049155 and WO2009/031043.

Staphylococcus epidermis

Clostridium perfringens or Clostridium botulinums

Legionella pneumophila

Coxiella burnetii

Brucella, such as B. abortus, B. canis, B. melitensis, B. neotomae, B.ovis, B. suis, B. pinnipediae.

Francisella, such as F. novicida, F. philomiragia, F. tularensis.

Neisseria gonorrhoeae

Treponema pallidum

Haemophilus ducreyi

Enterococcus faecalis or Enterococcus faecium

Staphylococcus saprophyticus

Yersinia enterocolitica

Mycobacterium tuberculosis

Rickettsia

Listeria monocytogenes

Vibrio cholerae

Salmonella typhi

Borrelia burgdorferi

Porphyromonas gingivalis

Klebsiella

In some embodiments the immunogen elicits an immune response against oneof these viruses:

Orthomyxovirus: Useful immunogens can be from an influenza A, B or Cvirus, such as the hemagglutinin, neuraminidase or matrix M2 proteins.Where the immunogen is an influenza A virus hemagglutinin it may be fromany subtype e.g. H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13,H14, H15 or H16.

Paramyxoviridae viruses: immunogens include, but are not limited to,those derived from Pneumoviruses (e.g. respiratory syncytial virus,RSV), Rubulaviruses (e.g. mumps virus), Paramyxoviruses (e.g.parainfluenza virus), Metapneumoviruses and Morbilliviruses (e.g.measles virus).

Poxviridae: immunogens include, but are not limited to, those derivedfrom Orthopoxvirus such as Variola vera, including but not limited to,Variola major and Variola minor.

Picornavirus: immunogens include, but are not limited to, those derivedfrom Picornaviruses, such as Enteroviruses, Rhinoviruses, Heparnavirus,Cardioviruses and Aphthoviruses. In one embodiment, the enterovirus is apoliovirus e.g. a type 1, type 2 and/or type 3 poliovirus. In anotherembodiment, the enterovirus is an EV71 enterovirus. In anotherembodiment, the enterovirus is a coxsackie A or B virus.

Bunyavirus: immunogens include, but are not limited to, those derivedfrom an Orthobunyavirus, such as California encephalitis virus, aPhlebovirus, such as Rift Valley Fever virus, or a Nairovirus, such asCrimean-Congo hemorrhagic fever virus.

Heparnavirus: immunogens include, but are not limited to, those derivedfrom a Heparnavirus, such as hepatitis A virus (HAV).

Filovirus: immunogens include, but are not limited to, those derivedfrom a filovirus, such as an Ebola virus (including a Zaire, IvoryCoast, Reston or Sudan ebolavirus) or a Marburg virus.

Togavirus: immunogens include, but are not limited to, those derivedfrom a Togavirus, such as a Rubivirus, an Alphavirus, or an Arterivirus.This includes rubella virus.

Flavivirus: immunogens include, but are not limited to, those derivedfrom a Flavivirus, such as Tick-borne encephalitis (TBE) virus, Dengue(types 1, 2, 3 or 4) virus, Yellow Fever virus, Japanese encephalitisvirus, Kyasanur Forest Virus, West Nile encephalitis virus, St. Louisencephalitis virus, Russian spring-summer encephalitis virus, Powassanencephalitis virus.

Pestivirus: immunogens include, but are not limited to, those derivedfrom a Pestivirus, such as Bovine viral diarrhea (BVDV), Classical swinefever (CSFV) or Border disease (BDV).

Hepadnavirus: immunogens include, but are not limited to, those derivedfrom a Hepadnavirus, such as Hepatitis B virus. A composition caninclude hepatitis B virus surface antigen (HBsAg).

Other hepatitis viruses: A composition can include an immunogen from ahepatitis C virus, delta hepatitis virus, hepatitis E virus, orhepatitis G virus.

Rhabdovirus: immunogens include, but are not limited to, those derivedfrom a Rhabdovirus, such as a Lyssavirus (e.g. a Rabies virus) andVesiculovirus (VSV).

Caliciviridae: immunogens include, but are not limited to, those derivedfrom Calciviridae, such as Norwalk virus (Norovirus), and Norwalk-likeViruses, such as Hawaii Virus and Snow Mountain Virus.

Coronavirus: immunogens include, but are not limited to, those derivedfrom a SARS coronavirus, avian infectious bronchitis (IBV), Mousehepatitis virus (MHV), and Porcine transmissible gastroenteritis virus(TGEV). The coronavirus immunogen may be a spike polypeptide.

Retrovirus: immunogens include, but are not limited to, those derivedfrom an Oncovirus, a Lentivirus (e.g. HIV-1 or HIV-2) or a Spumavirus.

Reovirus: immunogens include, but are not limited to, those derived froman Orthoreovirus, a Rotavirus, an Orbivirus, or a Coltivirus.

Parvovirus: immunogens include, but are not limited to, those derivedfrom Parvovirus B19.

Herpesvirus: immunogens include, but are not limited to, those derivedfrom a human herpesvirus, such as, by way of example only, HerpesSimplex Viruses (HSV) (e.g. HSV types 1 and 2), Varicella-zoster virus(VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV), HumanHerpesvirus 6 (HHV6), Human Herpesvirus 7 (HHV7), and Human Herpesvirus8 (HHV8).

Papovaviruses: immunogens include, but are not limited to, those derivedfrom Papillomaviruses and Polyomaviruses. The (human) papillomavirus maybe of serotype 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42,47, 51, 57, 58, 63 or 65 e.g. from one or more of serotypes 6, 11, 16and/or 18.

Adenovirus: immunogens include those derived from serotype 36 (Ad-36).

In some embodiments, the immunogen elicits an immune response against avirus which infects fish, such as: infectious salmon anemia virus(ISAV), salmon pancreatic disease virus (SPDV), infectious pancreaticnecrosis virus (IPNV), channel catfish virus (CCV), fish lymphocystisdisease virus (FLDV), infectious hematopoietic necrosis virus (IHNV),koi herpesvirus, salmon picorna-like virus (also known as picorna-likevirus of atlantic salmon), landlocked salmon virus (LSV), atlanticsalmon rotavirus (ASR), trout strawberry disease virus (TSD), cohosalmon tumor virus (CSTV), or viral hemorrhagic septicemia virus (VHSV).

Fungal immunogens may be derived from Dermatophytres, including:Epidermophyton floccusum, Microsporum audouini, Microsporum canis,Microsporum distortum, Microsporum equinum, Microsporum gypsum,Microsporum nanum, Trichophyton concentricum, Trichophyton equinum,Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini,Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophytonrubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophytonverrucosum, T. verrucosum var. album, var. discoides, var. ochraceum,Trichophyton violaceum, and/or Trichophyton faviforme; or fromAspergillus fumigatus, Aspergillus flavus, Aspergillus niger,Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowii,Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus,Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata,Candida krusei, Candida parapsilosis, Candida stellatoidea, Candidakusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis,Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis,Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum,Histoplasma capsulatum, Klebsiella pneumoniae, Microsporidia,Encephalitozoon spp., Septata intestinalis and Enterocytozoon bieneusi;the less common are Brachiola spp, Microsporidium spp., Nosema spp.,Pleistophora spp., Trachipleistophora spp., Vittaforma sppParacoccidioides brasiliensis, Pneumocystis carinii, Pythiumninsidiosum, Pityrosporum ovale, Sacharomyces cerevisae, Saccharomycesboulardii, Saccharomyces pombe, Scedosporium apiosperum, Sporothrixschenckii, Trichosporon beigelii, Toxoplasma gondii, Penicilliummarneffei, Malassezia spp., Fonsecaea spp., Wangiella spp., Sporothrixspp., Basidiobolus spp., Conidiobolus spp., Rhizopus spp, Mucor spp,Absidia spp, Mortierella spp, Cunninghamella spp, Saksenaea spp.,Alternaria spp, Curvularia spp, Helminthosporium spp, Fusarium spp,Aspergillus spp, Penicillium spp, Monolinia spp, Rhizoctonia spp,Paecilomyces spp, Pithomyces spp, and Cladosporium spp.

In some embodiments the immunogen elicits an immune response against aparasite from the Plasmodium genus, such as P. falciparum, P. vivax, P.malariae or P. ovale. Thus the invention may be used for immunisingagainst malaria. In some embodiments the immunogen elicits an immuneresponse against a parasite from the Caligidae family, particularlythose from the Lepeophtheirus and Caligus genera e.g. sea lice such asLepeophtheirus salmonis or Caligus rogercresseyi.

In some embodiments the immunogen elicits an immune response against:pollen allergens (tree-, herb, weed-, and grass pollen allergens);insect or arachnid allergens (inhalant, saliva and venom allergens, e.g.mite allergens, cockroach and midges allergens, hymenopthera venomallergens); animal hair and dandruff allergens (from e.g. dog, cat,horse, rat, mouse, etc.); and food allergens (e.g. a gliadin). Importantpollen allergens from trees, grasses and herbs are such originating fromthe taxonomic orders of Fagales, Oleales, Pinales and platanaceaeincluding, but not limited to, birch (Betula), alder (Alnus), hazel(Corylus), hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeria andJuniperus), plane tree (Platanus), the order of Poales including grassesof the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris,Secale, and Sorghum, the orders of Asterales and Urticales includingherbs of the genera Ambrosia, Artemisia, and Parietaria. Other importantinhalation allergens are those from house dust mites of the genusDermatophagoides and Euroglyphus, storage mite e.g. Lepidoglyphys,Glycyphagus and Tyrophagus, those from cockroaches, midges and flease.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides, and thosefrom mammals such as cat, dog and horse, venom allergens including suchoriginating from stinging or biting insects such as those from thetaxonomic order of Hymenoptera including bees (Apidae), wasps(Vespidea), and ants (Formicoidae).

In some embodiments the immunogen is a tumor antigen selected from: (a)cancer-testis antigens such as NY-ESO-1, SSX2, SCP1 as well as RAGE,BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1, GAGE-2,MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which canbe used, for example, to address melanoma, lung, head and neck, NSCLC,breast, gastrointestinal, and bladder tumors; (b) mutated antigens, forexample, p53 (associated with various solid tumors, e.g., colorectal,lung, head and neck cancer), p21/Ras (associated with, e.g., melanoma,pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g.,melanoma), MUM1 (associated with, e.g., melanoma), caspase-8 (associatedwith, e.g., head and neck cancer), CIA 0205 (associated with, e.g.,bladder cancer), HLA-A2-R1701, beta catenin (associated with, e.g.,melanoma), TCR (associated with, e.g., T-cell non-Hodgkins lymphoma),BCR-abl (associated with, e.g., chronic myelogenous leukemia),triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT; (c)over-expressed antigens, for example, Galectin 4 (associated with, e.g.,colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin'sdisease), proteinase 3 (associated with, e.g., chronic myelogenousleukemia), VVT 1 (associated with, e.g., various leukemias), carbonicanhydrase (associated with, e.g., renal cancer), aldolase A (associatedwith, e.g., lung cancer), PRAME (associated with, e.g., melanoma),HER-2/neu (associated with, e.g., breast, colon, lung and ovariancancer), mammaglobin, alpha-fetoprotein (associated with, e.g.,hepatoma), KSA (associated with, e.g., colorectal cancer), gastrin(associated with, e.g., pancreatic and gastric cancer), telomerasecatalytic protein, MUC-1 (associated with, e.g., breast and ovariancancer), G-250 (associated with, e.g., renal cell carcinoma), p53(associated with, e.g., breast, colon cancer), and carcinoembryonicantigen (associated with, e.g., breast cancer, lung cancer, and cancersof the gastrointestinal tract such as colorectal cancer); (d) sharedantigens, for example, melanoma-melanocyte differentiation antigens suchas MART-1/Melan A, gp100, MC1R, melanocyte-stimulating hormone receptor,tyrosinase, tyrosinase related protein-1/TRP1 and tyrosinase relatedprotein-2/TRP2 (associated with, e.g., melanoma); (e) prostateassociated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2,associated with e.g., prostate cancer; (f) immunoglobulin idiotypes(associated with myeloma and B cell lymphomas, for example). In certainembodiments, tumor immunogens include, but are not limited to, p15,Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virusantigens, EBNA, human papillomavirus (HPV) antigens, including E6 andE7, hepatitis B and C virus antigens, human T-cell lymphotropic virusantigens, TSP-180, p185erbB2, p180erbB-3, c-met, mn-23H1, TAG-72-4, CA19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4,791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29BCAA), CA195, CA 242, CA-50, CAM43, CD68KP1, CO-029, FGF-5, Ga733 (EpCAM),HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16,TA-90 (Mac-2 binding protein/cyclophilin C-associated protein), TAAL6,TAG72, TLP, TPS, and the like.

Pharmaceutical Compositions

A pharmaceutical composition of the invention, particularly one usefulfor immunization, may include one or more small moleculeimmunopotentiators. For example, the composition may include a TLR2agonist (e.g. Pam3CSK4), a TLR4 agonist (e.g. an aminoalkylglucosaminide phosphate, such as E6020), a TLR7 agonist (e.g.imiquimod), a TLR8 agonist (e.g. resiquimod) and/or a TLR9 agonist (e.g.IC31). Any such agonist ideally has a molecular weight of <2000 Da. Suchagonist(s) can, in some embodiments, be encapsulated with the RNA insideliposomes, or encapsulated or complexed with LNPs, but in otherembodiments they are unencapsulated or uncomplexed.

Pharmaceutical compositions of the invention may have an osmolality ofbetween 200 mOsm/kg and 400 mOsm/kg, e.g. between 240-360 mOsm/kg, orbetween 290-310 mOsm/kg.

Pharmaceutical compositions of the invention may include one or morepreservatives, such as thiomersal or 2 phenoxyethanol. Mercury-freecompositions can be made and preservative-free vaccines can be prepared.

Compositions comprise an immunologically effective amount of lipidcompositions described herein (e.g., liposomes and LNPs), as well as anyother components, as needed. Immunologically effective amount refers tothe amount administered to an individual, either in a single dose or aspart of a series, is effective for treatment (e.g., prophylactic immuneresponse against a pathogen). This amount varies depending upon thehealth and physical condition of the individual to be treated, age, thetaxonomic group of individual to be treated (e.g. non-human primate,primate, etc.), the capacity of the individual's immune system tosynthesise antibodies, the degree of protection desired, the formulationof the vaccine, the treating doctor's assessment of the medicalsituation, and other relevant factors. It is expected that the amountwill fall in a relatively broad range that can be determined throughroutine trials. The compositions of the invention will generally beexpressed in terms of the amount of RNA per dose. A preferred dose has≤100 μg RNA (e.g. from 10-100 μg, such as about 10 μg, 25 μg, 50 μg, 75μg or 100 μg), but expression can be seen at much lower levels e.g. ≤1μg/dose, ≤100 ng/dose, ≤10 ng/dose, ≤1 ng/dose, etc.

The invention also provides a delivery device (e.g. syringe, nebuliser,sprayer, inhaler, dermal patch, etc.) containing a pharmaceuticalcomposition of the invention. This device can be used to administer thecomposition to a vertebrate subject.

Liposomes or LNPs of the invention do not comprise ribosomes.

Methods of Treatment and Medical Uses

The liposome-formulated or LNP-formulated RNA and pharmaceuticalcompositions described herein are for in vivo use for inducing an immuneresponse against an immunogen of interest.

The invention provides a method for inducing an immune response in avertebrate comprising administering an effective amount of theliposome-formulated or LNP-formulated RNA, or pharmaceuticalcomposition, as described herein. The immune response is preferablyprotective and preferably involves antibodies and/or cell-mediatedimmunity. The compositions may be used for both priming and boostingpurposes. Alternatively, a prime-boost immunization schedule can be amix of RNA and the corresponding polypeptide antigen (e.g., RNA prime,protein boost).

The invention also provides a liposome, LNP, or pharmaceuticalcomposition for use in inducing an immune response in a vertebrate.

The invention also provides the use of a liposome, LNP, orpharmaceutical composition in the manufacture of a medicament forinducing an immune response in a vertebrate.

By inducing an immune response in the vertebrate by these uses andmethods, the vertebrate can be protected against various diseases and/orinfections e.g. against bacterial and/or viral diseases as discussedabove. The liposomes, LNPs, and compositions are immunogenic, and aremore preferably vaccine compositions. Vaccines according to theinvention may either be prophylactic (i.e. to prevent infection) ortherapeutic (i.e. to treat infection), but will typically beprophylactic.

The vertebrate is preferably a mammal, such as a human or a largeveterinary mammal (e.g. horses, cattle, deer, goats, pigs). As usedherein “large mammal” refers to mammals having a typical or averageadult weight of at least 5 kg, preferably at least 7 kg. Such largemammals can include, for example, humans, non-human primates, dogs,pigs, cattle, deer, goats, and is meant to exclude small mammals, suchas mice, rats, guinea pigs, and other rodents.

Where the vaccine is for prophylactic use, the human is preferably achild (e.g. a toddler or infant) or a teenager; where the vaccine is fortherapeutic use, the human is preferably a teenager or an adult. Avaccine intended for children may also be administered to adults e.g. toassess safety, dosage, immunogenicity, etc.

Vaccines prepared according to the invention may be used to treat bothchildren and adults. Thus a human patient may be less than 1 year old,less than 5 years old, 1-5 years old, 5-15 years old, 15-55 years old,or at least 55 years old. Preferred patients for receiving the vaccinesare the elderly (e.g. ≥50 years old, ≥60 years old, and preferably ≥65years), the young (e.g. ≤5 years old), hospitalised patients, healthcareworkers, armed service and military personnel, pregnant women, thechronically ill, or immunodeficient patients. The vaccines are notsuitable solely for these groups, however, and may be used moregenerally in a population. Compositions of the invention will generallybe administered directly to a patient. Direct delivery may beaccomplished by parenteral injection (e.g. subcutaneously,intraperitoneally, intravenously, intramuscularly, intradermally, or tothe interstitial space of a tissue; intraglossal injection is nottypically used for immunization purposes. Alternative delivery routesinclude rectal, oral (e.g. tablet, spray), buccal, sublingual, vaginal,topical, transdermal or transcutaneous, intranasal, ocular, aural,pulmonary or other mucosal administration. Intradermal and intramuscularadministration are two preferred routes. Injection may be via a needle(e.g. a hypodermic needle), but needle-free injection may alternativelybe used. A typical intramuscular dose is 0.5 ml.

The invention may be used to induce systemic and/or mucosal immunity,preferably to elicit an enhanced systemic and/or mucosal immunity.

Dosage can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunisation schedule and/or ina booster immunisation schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes e.g. a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Multiple doses will typically be administered at least 1 week apart(e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). In oneembodiment, multiple doses may be administered approximately 6 weeks, 10weeks and 14 weeks after birth, e.g. at an age of 6 weeks, 10 weeks and14 weeks, as often used in the World Health Organisation's ExpandedProgram on Immunisation (“EPI”). In an alternative embodiment, twoprimary doses are administered about two months apart, e.g. about 7, 8or 9 weeks apart, followed by one or more booster doses about 6 monthsto 1 year after the second primary dose, e.g. about 6, 8, 10 or 12months after the second primary dose. In a further embodiment, threeprimary doses are administered about two months apart, e.g. about 7, 8or 9 weeks apart, followed by one or more booster doses about 6 monthsto 1 year after the third primary dose, e.g. about 6, 8, 10, or 12months after the third primary dose.

EXAMPLES Cationic Lipids of Formula (I)

The following examples are intended to illustrate the invention and arenot to be construed as being limitations thereon. Temperatures are givenin degrees centigrade. If not mentioned otherwise, all evaporativeconcentrations are performed under reduced pressure, preferably betweenabout 15 mm Hg and 100 mm Hg (=20-133 mbar). The structure of finalproducts, intermediates and starting materials is confirmed by standardanalytical methods, e.g., microanalysis or spectroscopiccharacteristics, e.g., MS, IR, or NMR. Abbreviations used are thoseconventional in the art, some of which are defined below.

Flash column purification is preferably carried out on silica gel usingan appropriate eluent of isocratic or gradient composition.

HPLC analysis is performed on a Waters Atlantis dC18 column (4.6×150 mm,3 mm), with gradient elution (0% to 95% acetonitrile in water modifiedwith 0.1% v/v trifluoroacetic acid over 20 min and a flow rate of 1.4mL/min), unless otherwise described. 1H NMR spectra were recorded on aBruker Avance II 400 MHz spectrometer. All chemical shifts are reportedin parts per million (δ) relative to tetramethylsilane. The followingabbreviations are used to denote signal patterns: s=singlet, d=doublet,t=triplet, q=quartet, m=multiplet, br=broad. ES-MS data were recordedusing a Waters LTC Premier mass spectrometer with a dual electrosprayionization source on an Agilent 1100 liquid chromatograph.Sulfadimethoxine [Sigma, m/z=311.0814 (M+1)] was used as a referenceacquired through the LockSpray™ channel every third scan. The massaccuracy of the system has been found to be <5 ppm.

Abbreviations

-   AcOH acetic acid-   Aq aqueous-   Ar aryl-   Atm atmosphere-   BOC tert-Butyl-carbonate-   br.s., bs broad singlet-   ° C. Celsius-   CD₂Cl₂ deuterated dichloromethane-   CDCl₃ deuterated chloroform-   CH₂Cl₂, DCM dichloromethane-   CH₃CN, MeCN acetonitrile-   d doublet-   dd doublet of doublets-   ddd doublet of doublets of doublets-   DIPEA N-ethyldiisopropylamine-   DME 1,4-dimethoxyethane-   DMF N,N-dimethylformamide-   DMAP dimethyl aminopyridine-   DMSO dimethylsulfoxide-   dt doublet of triplets-   EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-   EtOAc ethyl acetate-   EtOH ethanol-   FCC flash column chromatography-   G gauge-   h hour-   HBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate-   HCl hydrochloric acid-   HMPA hexamethylphosphoramide-   HPLC high pressure liquid chromatography-   HT high throughput-   IBX 2-lodoxybenzoic acid-   i-PrOH isopropyl alcohol-   H₂O water-   K kelvin-   KOH potassium hydroxide-   LC liquid chromatography-   M molar-   m multiplet, mass-   MeOH methanol-   MgSO₄ magnesium sulfate-   MHz megahertz-   mL milliliter-   mm millimeter-   mmol millimole-   min. minute-   mRNA messenger ribonucleic acid-   MS mass spectroscopy-   mw microwave-   NaH sodium hydride-   NaHMDS sodium hexamethyldisilazane-   NaOEt sodium ethoxide-   NaOH sodium hydroxide-   Na₂SO₄ sodium sulfate-   NEt₃ triethylamine-   ng nanogram-   NH₃ ammonia-   NMR nuclear magnetic resonance-   quint. quintuplet-   Pd/C palladium on carbon-   ppt precipitate-   rbf round bottom flask-   Rf retardation factor-   rt room temperature-   Rt Retention time-   s singlet-   sat. saturated-   siRNA small interfering ribonucleic acid-   SM starting material-   t triplet-   TFA trifluoroacetic acid-   TH F tetrahydrofuran-   TLC thin layer chromatography-   UPLC ultra performance liquid chromatography-   wt weight-   μg microgram-   μL microliter-   IBX 2-iodobenzoic acid-   PMA phosphomolybdic acid-   PPTS pyridinium p-toluenesulfonate-   TBDPS tert-butyl diphenyl silyl-   TBDPSCl tert-butyl diphenyl silyl chloride-   TBS tert-butyl silyl

All compounds are named using AutoNom.

LC Specificity:

LC Method 1:

The retention times (Rt) were obtained on a Waters Acquity SDS systemwith an Acquity BEH 1.7 μm 2.1×50 mm column. A gradient of H₂O (+0.1%formic acid)/CH₃CN (+0.1% formic acid) 60/40 to 0.1/99.9 was appliedover 1.4 min., then held for 0.6 min. (1.0 mL/min. as solvent flow) atan oven temperature of 50° C.

LC Method 2:

The retention times (Rt) were obtained on a Waters Acquity SDS systemwith an Acquity BEH 1.7 μm 2.1×50 mm column. A gradient of H₂O (+0.1%formic acid)/CH₃CN (+0.1% formic acid) 60/40 to 0.1/99.9 was appliedover 3.4 min., then held for 1.6 min. (1.0 mL/min. as solvent flow) atan oven temperature of 50° C.

LC Method 3:

The retention times (Rt) were obtained on an Agilent 1100 system with anInertsil C8 Column, 3.0 μm, 3.0×30 mm. A gradient of H₂O (+0.1% formicacid)/CH₃CN (+0.1% formic acid) 60/40 to 5/95 was applied over 1.0 min.,then held for 1.0 min. (2.0 mL/min. as solvent flow) at an oventemperature of 50° C.

LC Method 4:

The retention times (Rt) were obtained on a Waters Acquity SDS systemwith an Acquity BEH 1.7 μm 2.1×50 mm column. A gradient of H₂O (+0.1%formic acid)/CH₃CN (+0.1% formic acid) 45/55 to 0/100 was applied over2.0 min., then held for 3.0 min. (1.0 mL/min. as solvent flow) at anoven temperature of 50° C.

LC Method 5:

The retention times (Rt) were obtained on a Waters Acquity SDS systemwith an Acquity BEH 1.7 μm 2.1×50 mm column. A gradient of H₂O (+0.1%formic acid)/CH₃CN (+0.1% formic acid) 45/55 to 0/100 was applied over1.0 min., then held for 1.0 min. (1.0 mL/min. as solvent flow) at anoven temperature of 50° C.

LC Method 6:

The retention times (Rt) were obtained on an Agilent 1100 system on anInertsil C8 Column, 3.0 μm, 3.0×30 mm. A gradient of H₂O (+0.1% formicacid)/CH₃CN (+0.1% formic acid) 60/40 to 5/95 was applied over 1.0 min.,then held for 1.0 min. (2.0 mL/min. as solvent flow) at an oventemperature of 50° C.

LC Method 7:

The retention times (Rt) were obtained on an Agilent 1100 system with anXBridge C8 Column, 3.0 μm, 3.0×30 mm. A gradient of H₂O (+0.1% formicacid)/CH₃CN (+0.1% formic acid) 60/40 to 5/95 was applied over 1.0 min.,then held for 1.0 min. (2.0 mL/min. as solvent flow) at an oventemperature of 50° C.

LC Method 8:

The retention times (Rt) were obtained on an Agilent 1100 system with anXBridge C8 Column, 3.0 μm, 3.0×30 mm. A gradient of H₂O (+5 mM AmmoniumFormate, 2% ACN)/CH₃CN 60/40 to 5/95 was applied over 1.0 min., thenheld for 1.0 min. (2.0 mL/min. as solvent flow) at an oven temperatureof 50° C.

LC Method 9:

The retention times (Rt) were obtained on an Agilent 1100 system with anAtlantis C18 Column, 3.5 μm, 3.0×30 mm. A gradient of H₂O (+0.05%trifluoroacetic acid)/CH₃CN 60/40 to 2/98 was applied over 1.7 min.,then held for 0.3 min. (2.0 mL/min. as solvent flow), then changed to60/40 over 0.1 min at an oven temperature of 40° C.

LC Method 10:

The retention times (Rt) were obtained on an Agilent 1100 system with anXBridge C18 Column, 3.5 μm, 2.1×50 mm. A gradient of H₂O (+0.1% formicacid)/CH₃CN 95/5 to 5/95 was applied over 1.7 min., then held for 0.2min. (2.0 mL/min. as solvent flow), then changed to 95/5 over 0.1 min atan oven temperature of 40° C.

LC Method 11:

The retention times (Rt) were obtained on an Agilent 1100 system with anXBridge C18 Column, 3.5 μm, 2.1×50 mm. A gradient of H₂O (+0.1% formicacid)/CH₃CN 95/5 to 5/95 was applied over 1.7 min., then held for 0.2min. (2.0 mL/min. as solvent flow), then changed to 95/5 over 0.1 min atan oven temperature of 40° C.

LC method 12:

The retention times (Rt) were obtained on a Waters Acquity SDS systemwith an Acquity BEH C18 1.7 μm 2.1×50 mm column. A gradient of H₂O(+0.1% formic acid)/CH₃CN (+0.1% formic acid) 45/55 to 1/99 was appliedover 1.4 min., then a gradient of 1/99 to 0/100 was applied over 3.7min. (1.0 mL/min. as solvent flow) at an oven temperature of 50° C.

LC method 13:

The retention times (Rt) were obtained on a Waters Acquity SDS systemwith an Acquity BEH 1.7 μm 2.1×50 mm column. A gradient of H₂O (+0.1%formic acid)/CH₃CN (+0.1% formic acid) 60/40 to 2/98 was applied over3.4 min., then held for 1.7 min. (1.0 mL/min. as solvent flow) at anoven temperature of 50° C.

LC method 14:

The retention times (Rt) were obtained on a Waters Acquity SDS systemwith an Acquity BEH 1.7 μm 2.1×50 mm column. A gradient of H₂O (+0.1%formic acid)/CH₃CN (+0.1% formic acid) 45/55 to 1/99 was applied over0.7 min., then held for 1.3 min. (1.0 mL/min. as solvent flow) at anoven temperature of 50° C.

Synthesis of Example 1:2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azaoctadecan-18-yl)propane-1,3-diyldioctanoate Intermediate 1a: 5-(benzyloxy)pentyl methanesulfonate

Et₃N (10.79 mL, 78 mmol) was added in one portion via syringe to asolution of 5-Benzyloxy-1-pentanol (10.08 g, 51.9 mmol) in DCM (75 mL)in a round bottom flask charged with a magnetic stir bar at 0° C. underN₂. Next, MsCl (4.85 mL, 62.3 mmol) was added dropwise via syringe in 4separate portions at a rate such that the internal temperature did notexceed 15° C. The reaction was allowed to continue to stir for 1 hour,after which it was diluted with H₂O (200 mL) and DCM (150 mL). Theorganic layer was separated, and the aqueous layer was washed with DCM(225 mL). The combined organic layers were washed with brine (200 mL),dried with Na₂SO₄, filtered, and concentrated under reduced pressure toprovide the title compound as a crude orange oil (14.46 g, 99%). MS(M+1)=272.9, Rt=1.33 min (LC method 10).

Intermediate 1b: diethyl 2-(5-(benzyloxy)pentyl)malonate

To a cold, 0° C., solution of diethyl malonate (7.5 g, 46.8 mmol) in dryDMF (100 mL) under N₂ atmosphere was added 60% NaH (2.23 g, 56.2 mmol)portionwise over 10 min. The evolution of gas was observed. The mixturewas stirred at 0° C. for 30 min then Intermediate 1a (14.46 g, 51.5mmol) in dry DMF (41 mL) was added dropwise over 10 min followed bytetrabutylammonium iodide (1.73 g, 4.68 mmol). The mixture was thenheated at 100° C. for 1.5 h. The mixture was cooled at rt and leftovernight. The mixture was then quenched with sat. NH₄Cl (50 mL) anddiluted with H₂O (100 mL). The mixture was extracted with EtOAc (2×200mL). The combined organic layers were washed with H₂O (100 mL), brine(100 mL), dried over MgSO₄, filtered, and concentrated on vacuo. Theresidue was purified by silica gel column chromatography eluting with0-30% EtOAc/heptane to afford the title compound as an oil (11.7 g,74%). MS (M+1)=336.6, Rt=1.64 min (LC method 10).

Intermediate 1c: 2-(5-(benzyloxy)pentyl)propane-1,3-diol

To a cold solution, 0° C., of Intermediate 1b (5.5 g, 16.35 mmol) in dryTHF (20 mL) under N₂ was added 2.0 M LiAlH₄ in THF (24.52 mL, 49.0 mmol)dropwise over 15 min. The mixture was allowed to warm to rt and stirredovernight. The mixture was cooled to 0° C., treated again with 2.0 MLiAlH₄ in THF (16.35 mL, 32.7 mmol), allowed to warm to rt, and stirredover the weekend. The reaction was cooled to 0° C. and quenched withEtOAc (9.30 mL) dropwise over 10 min. The mixture was then treated withH₂O (3.10 mL) dropwise, a 15% NaOH solution (3.10 mL) solution dropwise,and then additional H₂O (9.30) dropwise. The mixture was stirred for 30min at rt. The mixture was filtered through a pad of Celite. The Celitewas washed with EtOAc. The filtrate was concentrated under reducedpressure to afford the title compound as a semi-wax solid (1.52 g, 37%).MS (M+1)=253.0, Rt=1.37 min (LC method 10).

Intermediate 1d: 2-(5-(benzyloxy)pentyl)propane-1,3-diyl dioctanoate

Pyridine (1.21 mL, 14.96 mmol) was added dropwise via syringe over ˜30seconds to a solution of Intermediate 1c (1.51 g, 5.98 mmol) in DCM (20mL) in a round bottom flask charged with a magnetic stir bar at 0° C.under N₂. Next, octanoyl chloride (2.145 mL, 12.57 mmol) was addeddropwise via syringe over several minutes, and the reaction was allowedto warm to rt and stirred overnight. The mixture was diluted with sat.NH₄Cl (100 mL) and CH₂Cl₂ (100 mL). The organic was separated. Theaqueous was extracted with CH₂Cl₂ (100 mL). The combined organics weredried over MgSO₄, filtered, and concentrated under reduced pressure. Thecrude material was purified by silica gel column chromatography elutingwith 0-10% EtOAc/heptane to afford the title compound as a colorless oil(2.88 g, 95%). MS (M+1)=505.6, Rt=1.77 min (LC method 1).

Intermediate 1e: 2-(5-hydroxypentyl)propane-1,3-diyl dioctanoate

To a solution of Intermediate Id (2.5 g, 4.95 mmol) in MeOH (25 ml) atrt was added 10% Pd/C, wet degussa type (264 mg). The mixture wasstirred under a H₂ balloon overnight. The crude reaction mixture wasfiltered through a pad of celite and filtrate was concentrated underreduced pressure to afford the title compound as a colorless oil (2.0 g,97%). ¹H NMR (400 MHz, CD₂Cl₂) δ 4.11-3.96 (m, 4H), 3.59 (t, J=6.5 Hz,2H), 2.28 (t, J=7.5 Hz, 4H), 2.04-1.91 (m, 1H), 1.66-1.48 (m, 7H),1.41-1.34 (m, 6H), 1.34-1.20 (m, 16H), 0.88 (t, J=6.8 Hz, 6H).

Intermediate 1f: 7-(octanoyloxy)-6-((octanoyloxy)methyl)heptanoic acid

TEMPO (0.151 g, 0.965 mmol) was added in one portion to Intermediate 1e(2.0 g, 4.82 mmol) in MeCN:H₂O (46.84 mL, 1:1 ratio) in a vial chargedwith a magnetic stir bar at rt. Next, iodobenzene diacetate (3.42 g,10.61 mmol) was added in one portion, and the reaction was allowed tocontinue to stir at rt overnight, after which the reaction was quenchedwith 15% aqueous sodium thiosulfate (50 mL). The reaction was dilutedwith H₂O and extracted with EtOAc (3×100 mL). The combined organiclayers were dried with Na₂SO₄, filtered, and concentrated under reducedpressure to provide a pale yellow oil. The oil was dissolved in toluene(15 mL) and concentrated under reduced pressure (×6) to provide a paleyellow oil, which was dissolved in DCM and concentrated under reducedpressure (×3) to provide the title compound (plus minor aromaticimpurities which could correspond to either residual toluene oriodobenzene) as a pale yellow oil (2.0 g, 97%). MS (M−1)=427.2, Rt=1.73min (LC method 9).

Intermediate 1g: 3-((tert-butyldimethylsilyl)oxy)propanal

In a 1 L round-bottom flask equipped with a stir bar,Tertbutyldimethylsilyloxypropanol (20 g, 105 mmol) was dissolved in DCM(500 mL). Et₃N (43.9 mL, 315 mmol) was added. In a second 500 mL flaskequipped with a stirbar, SO₃.Py (25.08 g, 158 mmol) was dissolved inDMSO (100 mL, 1409 mmol). The resulting solution was added dropwise tothe alcohol solution at 0° C. (in an ice-water bath). The reaction wasstirred while warming to rt over the weekend. Water and DCM were addedto the mixture in a separatory funnel. The organics were then washedwith water, extracted in DCM, dried over MgSO₄, filtered andconcentrated (cold) under reduced pressure to give crude productmixture. Purification by silica gel column chromatography (330 g column,100% DCM) provided the title compound (17.7 g, 89%). ¹H NMR (400 MHz,CDCl₃) δ=9.81 (t, J=2.1 Hz, 1H), 3.99 (t, J=6.0 Hz, 2H), 2.61 (td,J=6.0, 2.3 Hz, 2H), 0.88 (s, 9H), 0.07 (s, 6H).

Intermediate 1h: 1-((tert-butyldimethylsilyl)oxy)pentadecan-3-ol

In a 500 mL round-bottom flask, Intermediate 1g (17.7 g, 94 mmol) wasdissolved in THF (100 mL), and cooled to 0° C. in an ice-water bath.Dodecylmagnesiumbromide, 1M in diethyl ether (132 mL, 132 mmol) was thenadded to the aldehyde dropwise over 10 min via pipette, the ice-bath wasremoved, and reaction was stirred at rt for 30 min. The reaction flaskwas cooled again to 0° C. in an ice-bath. Sat. NH₄Cl soln was addedslowly to adjust to pH ˜7 (600 mL), and the mixture was poured into a 1L separatory funnel. The organics were then washed with sat. ammoniumchloride solution, extracted in EtOAc, dried over MgSO₄, filtered andconcentrated under reduced pressure to give the crude product mixture.Purification by silica gel column chromatography (330 g column, 100%Heptanes for 2 column volumes, 0% to 2% EtOAc/Heptane for 1 columnvolume, 2% EtOAc/Heptane for 3 column volumes, 2% to 5% EtOAc/Heptanefor 1 column volume, then 5% EtOAc/Heptane for 10 column volumes)provided the title compound (24.9 g, 74%). ¹H NMR (400 MHz, CDCl₃) δ3.97-3.87 (m, 1H), 3.87-3.77 (m, 2H), 1.69-1.60 (m, 2H), 1.57-1.36 (m,3H), 1.36-1.19 (br, 19H), 0.93-0.84 (m, 12H), 0.09 (s, 6H).

Intermediate 1i: 1-((tert-butyldimethylsilyl)oxy)pentadecan-3-yl(4-nitrophenyl) carbonate

4-nitrophenyl carbonochloridate (2.084 g, 9.92 mmol) was added in oneportion to a solution of Intermediate 1h (2.9660 g, 8.27 mmol) in DCM(28 mL) in a round bottom flask charged with a magnetic stir bar at rt.The reaction was fitted with a septum and placed under N₂, after whichpyridine (1.003 mL, 12.40 mmol) was added dropwise via syringe overseveral minutes. The reaction was allowed to stir at rt overnight. After24 hours of reaction time, the reaction was diluted with H₂O (100 mL)and DCM (125 mL). The organic layer was separated, and the aqueous layerwas washed with DCM (125 mL). The combined organic layers were washedwith brine (100 mL), dried with Na₂SO₄, filtered, and concentrated underreduced pressure to provide an off white residue. The crude residue waspurified by silica gel column chromatography (80 g column, liquidloading, 0-2.5% EtOAc:heptane) to provide 3.144 g (73%) of the titlecompound (plus minor unidentified impurity peaks) as a colorless oil. ¹HNMR (400 MHz, CD₂Cl₂) δ 8.29-8.24 (m, 2H), 7.41-7.35 (m, 2H), 5.03-4.95(m, 1H), 3.73 (dd, J=6.6, 5.7 Hz, 2H), 1.95-1.80 (m, 2H), 1.80-1.63 (m,2H), 1.45-1.19 (m, 20H), 0.92-0.83 (m, 12H), 0.06 (s, 6H).

Intermediate 1j: 1-((tert-butyldimethylsilyl)oxy)pentadecan-3-yl(3-(diethylamino)propyl) carbonate

3-(diethylamino)propan-1-ol (3.48 mL, 22.26 mmol) was added dropwise viasyringe over a few minutes to Intermediate 1i (2.9153 g, 5.57 mmol) inDCM (40 mL) in a round bottom flask charged with a magnetic stir barunder N₂. Next, pyridine (2.251 mL, 27.8 mmol) was added dropwise viasyringe over 30 seconds, followed by the addition of DMAP (0.136 g,1.113 mmol) in one portion. The reaction was allowed to continue to stirat rt overnight. After 22 hours of reaction time, the reaction wasdiluted with H₂O (200 mL) and DCM (200 mL). The organic layer wasseparated and the aqueous layer was washed with DCM (200 mL). Thecombined organic layers were washed with brine (100 mL), dried withNa₂SO₄, filtered, and concentrated under reduced pressure to provide acrude yellow-orange oil, which was purified by silica gel columnchromatography (120 g column, liquid loading (in DCM), 0-8% MeOH:DCM) toprovide a yellow oil. The yellow oil was passed through a bond elut NH2column (10 g), eluting with DCM. The filtrate was concentrated underreduced pressure to provide a pale yellow oil. The bond elut procedurewas repeated again to provide the title compound as a colorless oil(2.0887 g, 73%). MS (M+1)=516.4, Rt=1.42 min (LC method 9).

Intermediate 1k: 3-(diethylamino)propyl (1-hydroxypentadecan-3-yl)carbonate

To a solution of Intermediate 1j (2.2 g, 4.26 mmol) in MeOH (50 mL) atrt was added CAN (5.14 g, 9.38 mmol) in one portion. The mixture wasstirred at rt and followed by TLC (70% EtOAc/heptane, KMNO₄ stain).After the consumption of starting material was observed, the mixture wasdiluted with CH₂Cl₂ (100 mL), and washed with sat. NaHCO₃ (2×100 mL).The aqueous layer was extracted with CH₂Cl₂ (100 mL). The combinedorganics were dried over Na₂SO₄, filtered, and concentrated underreduced pressure to afford the title compound (along with some minorimpurities) as an oil. MS (M+1)=402.2, Rt=0.76 min (LC Method 9).

Preparation of Final Compounds

Example 1: Synthesis of2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azaoctadecan-18-yl)propane-1,3-diyldioctanoate

In a round bottom flask, Intermediate 1f (1.99 g, 4.66 mmol), DMAP(0.207 g, 1.693 mmol), DIPEA (1.48 mL, 8.47 mmol), and Intermediate 1k(1.7 g, 13.07 mmol) were taken into dichloromethane (20 mL). EDC.HCl(1.62 g, 8.47 mmol) was added in one portion, and the reaction wasstirred at ambient temperature. After 24 h, the reaction wasconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (0% to 5% MeOH in CH₂Cl₂). Fractions werecollected and solvents were removed under reduced pressure to provide ayellow oil (3.8 g). The oil was repurified by silica gel columnchromatography (0-75% EtOAc/heptane) to afford the title compound as anoff-white oil (2.6 g, 71.8%). ¹H NMR (400 MHz, CDCl₃) δ=4.80 (t, J=6.1Hz, 1H), 4.25-3.96 (m, 8H), 2.62 (br. s., 4H), 2.30 (t, J=7.5 Hz, 6H),2.05-1.85 (m, 5H), 1.71-1.53 (m, 8H), 1.43-1.21 (m, 42H), 1.21-0.98 (m,6H), 0.96-0.79 (m, 9H). ¹³C NMR (101 MHz, CDCl₃) δ=174.15 (s, 2C),173.71, 155.20, 75.90, 66.41, 64.14 (s, 2C), 60.85, 49.32, 47.10 (s,2C), 37.46, 34.56 (s, 3C), 34.52, 34.29, 33.28, 32.22, 31.97 (s, 2C),29.95 (s, 2C), 29.88, 29.80, 29.76, 29.66, 29.41 (s, 2C), 29.23 (s, 2C),28.20, 26.64, 25.36, 25.25 (s, 4C), 23.00, 22.90 (s, 2C), 14.44, 14.38(s, 2C), 11.37 (s, 2C).

The following examples can be prepared using similar coupling methods tothose employed for the synthesis of Example 1.

Example 2:2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2-azaheptadecan-17-yl)propane-1,3-diyldioctanoate

¹H NMR (400 MHz, CDCl₃) δ=4.80 (t, J=5.4 Hz, 1H), 4.28-3.98 (m, 8H),2.65-2.35 (m, 8H), 2.30 (t, J=7.5 Hz, 6H), 2.09-1.84 (m, 5H), 1.74-1.50(m, 8H), 1.48-1.33 (m, 8H), 1.33-1.18 (m, 32H), 0.88 (t, J=6.8 Hz, 9H).¹³C NMR (101 MHz, CDCl₃) δ=173.47 (2C), 173.06, 154.53, 75.34, 63.55(3C), 60.18, 55.48, 44.41 (2C), 36.90, 33.95 (2C), 33.91, 33.68, 32.70,31.59, 31.34 (2C), 29.33 (2C), 29.31, 29.24, 29.17, 29.12, 29.02, 28.79(2C), 28.59 (2C), 27.61, 26.02 (2C), 24.73, 24.64 (3C), 22.36, 22.26(2C), 13.78, 13.73 (2C).

Example 3:2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2-azapentadecan-15-yl)propane-1,3-diyldioctanoate

¹H NMR (400 MHz, CDCl₃) δ=4.88-4.71 (m, 1H), 4.28-3.98 (m, 8H), 2.54(br. s., 2H), 2.48-2.35 (m, 6H), 2.31 (t, J=7.7 Hz, 6H), 2.13-1.98 (m,2H), 1.98-1.85 (m, 3H), 1.71 (q, J=7.5 Hz, 2H), 1.65-1.49 (m, 6H),1.40-1.16 (m, 36H), 0.97-0.80 (m, 9H). ¹³C NMR (101 MHz, CDCl₃) δ=173.69(2C), 172.89, 154.84, 75.60, 63.55 (3C), 60.72, 55.77, 45.05-44.29 (2C),36.80, 34.24, 34.20 (2C), 33.00, 31.90, 31.64 (2C), 31.42, 29.65, 29.63(2C), 29.56, 29.49, 29.44, 29.33, 29.09 (2C), 28.90 (2C), 25.06, 24.91(3C), 23.44, 22.66, 22.58 (2C), 14.10, 14.04 (2C).

Example 4:2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azahexadecan-16-yl)propane-1,3-diyldioctanoate

¹H NMR (400 MHz, CDCl₃) δ=4.80 (t, J=6.0 Hz, 1H), 4.28-3.98 (m, 8H),2.63 (br. s., 6H), 2.40 (t, J=7.7 Hz, 2H), 2.31 (t, J=7.5 Hz, 4H),2.11-2.00 (m, 1H), 2.00-1.84 (m, 4H), 1.71 (q, J=7.5 Hz, 2H), 1.66-1.49(m, 6H), 1.40-1.21 (m, 36H), 1.09 (br. s., 6H), 0.88 (t, J=6.8 Hz, 9H).¹³C NMR (101 MHz, CDCl₃) δ=173.73 (2C), 172.89, 154.92, 75.44, 66.31(2C), 63.50 (2C), 60.79, 48.99, 46.82 (2C), 36.71, 34.22, 34.18 (2C),32.95, 31.89, 31.63 (2C), 31.37, 29.62 (3C), 29.55, 29.48, 29.45, 29.34,29.08 (2C), 28.90 (2C), 25.04, 24.89 (2C), 23.37, 22.67, 22.58 (2C),14.11, 14.05 (2C), 11.33 (2C).

Example 5:2-(8-dodecyl-2-methyl-6,12-dioxo-5,7,11-trioxa-2-azaheptadecan-17-yl)propane-1,3-diyldioctanoate

¹H NMR (400 MHz, CDCl₃) δ=4.91-4.71 (m, 1H), 4.29-4.18 (m, 2H),4.18-3.96 (m, 6H), 2.60 (t, J=5.8 Hz, 2H), 2.39-2.23 (m, 10H), 2.05-1.85(m, 3H), 1.71-1.51 (m, 8H), 1.42-1.19 (m, 44H), 0.96-0.82 (m, 9H). ¹³CNMR (101 MHz, CDCl₃) δ=173.81 (2C), 173.50, 155.02, 75.68, 65.46, 63.96(2C), 60.60, 57.63, 45.69 (2C), 37.29, 34.27 (2C), 34.20, 34.10, 33.01,31.91, 31.66 (2C), 29.65, 29.63 (2C), 29.55, 29.49, 29.44, 29.34, 29.26,29.11 (2C), 28.91 (2C), 28.08, 26.50, 25.02, 24.96 (2C), 24.72, 22.68,22.58 (2C), 14.10, 14.05 (2C).

Example 6:2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azanonadecan-19-yl)propane-1,3-diyldioctanoate

¹H NMR (400 MHz, CDCl₃) δ=4.89-4.72 (m, 1H), 4.26-3.96 (m, 8H),2.62-2.40 (m, 6H), 2.40-2.21 (m, 6H), 2.04-1.87 (m, 3H), 1.87-1.74 (m,2H), 1.74-1.52 (m, 8H), 1.41-1.17 (m, 42H), 1.01 (t, J=7.2 Hz, 6H),0.96-0.80 (m, 9H). ¹³C NMR (101 MHz, CDCl₃) δ=173.86 (2C), 173.54,154.99, 75.49, 66.57, 63.92 (2C), 60.61, 49.03, 46.89 (2C), 37.22, 34.26(2C), 34.20, 34.08, 32.98, 31.91, 31.66 (2C), 29.65, 29.63 (2C), 29.56,29.49, 29.45, 29.35, 29.25, 29.10 (2C), 28.92 (2C), 28.02, 26.49, 26.47,25.04, 24.94 (2C), 24.70, 22.68, 22.59 (2C), 14.13, 14.07 (2C), 11.78(2C).

Example 7:2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2-azaoctadecan-18-yl)propane-1,3-diyldioctanoate

¹H NMR (400 MHz, CDCl₃) δ 4.80 (t, J=6.15 Hz, 1H), 3.97-4.27 (m, 8H),2.46 (t, J=7.28 Hz, 2H), 2.18-2.39 (m, 12H), 1.83-2.05 (m, 5H),1.53-1.72 (m, 8H), 1.19-1.44 (m, 42H), 0.88 (t, J=6.65 Hz, 9H). ¹³C NMR(101 MHz, CDCl₃) δ=173.86 (2C), 173.55, 154.89, 75.60, 66.10, 63.90(2C), 60.54, 55.70, 44.89 (2C), 37.21, 34.25 (2C), 34.19, 34.08, 32.95,31.90, 31.65 (2C), 29.62 (2C), 29.56, 29.48, 29.44, 29.34, 29.24, 29.09(2C), 28.91 (2C), 28.01 (2C), 26.49, 26.39, 25.03, 24.94 (2C), 24.70,22.68, 22.59, 14.12, 14.07 (2C).

Example 8:2-(8-dodecyl-2-methyl-6,12-dioxo-5,7,11-trioxa-2-azaoctadecan-18-yl)propane-1,3-diyldioctanoate

¹H NMR (400 MHz, CDCl₃) Q=4.95-4.70 (m, 1H), 4.28-4.17 (m, 2H),4.17-3.96 (m, 6H), 2.60 (t, J=5.8 Hz, 2H), 2.36-2.24 (m, 12H), 2.03-1.86(m, 3H), 1.72-1.49 (m, 8H), 1.44-1.18 (m, 44H), 0.97-0.80 (m, 9H). ¹³CNMR (101 MHz, CDCl₃) δ=173.50 (2C), 173.28, 154.71, 75.38, 65.17, 63.67(2C), 60.26, 57.32, 45.40 (2C), 36.99, 33.97 (2C), 33.87, 33.84, 32.69,31.59, 31.34 (2C), 29.33, 29.31 (2C), 29.23, 29.17, 29.12 (2C), 29.02,28.79 (2C), 28.71, 28.59 (2C), 27.87, 26.32, 24.70, 24.65 (2C), 24.51,22.36, 22.26 (2C), 13.78, 13.73 (2C).

Example 9:2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azaicosan-20-yl)propane-1,3-diyldioctanoate

¹H NMR (400 MHz, CDCl₃) δ=4.92-4.73 (m, 1H), 4.26-3.97 (m, 8H), 2.52 (d,J=6.6 Hz, 6H), 2.38-2.23 (m, 6H), 2.02-1.88 (m, 3H), 1.88-1.74 (m, 2H),1.69-1.53 (m, 8H), 1.41-1.19 (m, 44H), 1.02 (t, J=7.1 Hz, 6H), 0.94-0.80(m, 9H). ¹³C NMR (101 MHz, CDCl₃) δ=173.81 (2C), 173.59, 155.01, 75.55,66.55, 63.99 (2C), 60.59, 49.10, 46.94 (2C), 37.31, 34.29 (2C), 34.22,34.16, 33.03, 31.91, 31.66 (2C), 29.65, 29.63 (2C), 29.56, 29.49, 29.44(2C), 29.34, 29.11 (2C), 29.03, 28.91 (2C), 28.19, 26.64, 26.53, 25.04,24.97 (2C), 24.83, 22.68, 22.58 (2C), 14.10, 14.05 (2C), 11.80 (2C).

Example 10:2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2-azanonadecan-19-yl)propane-1,3-diyldioctanoate

¹H NMR (400 MHz, CDCl₃) δ=4.95-4.72 (m, 1H), 4.28-3.98 (m, 8H),2.48-2.26 (m, 8H), 2.23 (s, 6H), 2.02-1.89 (m, 3H), 1.89-1.78 (m, 2H),1.69-1.58 (m, 8H), 1.41-1.19 (m, 44H), 0.96-0.79 (m, 9H). ¹³C NMR (101MHz, CDCl₃) δ=173.82 (2C), 173.60, 154.97, 75.60, 66.34, 63.99 (2C),60.59, 56.00, 45.46 (2C), 37.31, 34.29 (2C), 34.21, 34.16, 33.02, 31.91,31.66 (2C), 29.65, 29.63 (2C), 29.55, 29.49, 29.44 (2C), 29.34, 29.11(2C), 29.03, 28.91 (2C), 28.19, 26.99, 26.64, 25.05, 24.97 (2C), 24.83,22.68, 22.58 (2C), 14.10, 14.05 (2C).

Synthesis of Example 11 Intermediate 11a: 4,4-bis(octyloxy)butanenitrile

To a mixture of 4,4-diethoxybutanenitrile (15 g, 95 mmol) and octanol(37.3 g, 286 mmol) was added pyridinium p-toluenesulfonate (1.2 g, 4.77mmol) and the mixture was heated in a 105° C. bath. After 72 h, thereaction mixture was cooled to ambient temperature and purified onsilica gel using ethyl acetate/heptane as eluent to provide 9.34 g ofthe expected product. ¹H NMR (400 MHz, CDCl₃): δ=4.56 (t, J=5.40 Hz,1H), 3.61 (dt, J=9.16, 6.59 Hz, 2H), 3.44 (dt, J=9.22, 6.68 Hz, 2H),2.43 (t, J=7.28 Hz, 2H), 1.95 (td, J=7.34, 5.40 Hz, 2H), 1.50-1.66 (m,4H), 1.17-1.44 (m, 20H), 0.80-0.95 (m, 6H).

Intermediate 11b: 4,4-bis(octyloxy)butanoic acid

In a high pressure reaction vessel, Intermediate 11a (9.34 g, 28.7 mmol)is dissolved in 30 mL EtOH. KOH (4.83 g) was dissolved in 30 mL waterand the KOH solution was added to the EtOH solution. The tube was sealedand heated in a 1100C bath overnight. The mixture was cooled and dilutedwith EtOAc. 1 N HCl was added to adjust pH to 5, and the aqueous phasewas extracted with EtOAc twice. The combined organic extracts were driedover MgSO4, filtered, and concentrated under reduced pressure to provide10.9 g of the expected product. ¹H NMR (400 MHz, CDCl₃): δ=4.46 (t,J=5.52 Hz, 1H), 3.46-3.59 (m, 2H), 3.08-3.46 (m, 3H), 2.18 (t, J=7.28Hz, 2H), 1.72-1.89 (m, 2H), 1.46-1.63 (m, 4H), 1.28 (d, J=3.76 Hz, 20H),0.79-0.96 (m, 6H).

Preparation of Final Compounds

The following examples (Examples 11-15) can be prepared using similarmethods to those employed for the synthesis of Example 1.

Example 11: 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl4,4-bis(octyloxy)butanoate

¹H NMR (400 MHz, CDCl₃) δ=4.89-4.72 (m, 1H), 4.50 (t, J=5.6 Hz, 1H),4.28-4.09 (m, 4H), 3.68-3.48 (m, 2H), 3.41 (td, J=6.8, 9.2 Hz, 2H),2.61-2.45 (m, 6H), 2.39 (t, J=7.5 Hz, 2H), 1.99-1.88 (m, 4H), 1.81(quin, J=7.0 Hz, 2H), 1.72-1.47 (m, 8H), 1.42-1.20 (m, 38H), 1.02 (t,J=7.2 Hz, 6H), 0.94-0.82 (m, 9H). ¹³C NMR (101 MHz, CDCl₃) δ=173.27,154.98, 102.03, 75.50, 66.57, 66.07 (2C), 60.73, 49.03, 46.89 (2C),34.21, 32.96, 31.91, 31.83 (2C), 29.84 (2C), 29.64 (3C), 29.56, 29.50,29.44 (2C), 29.36 (3C), 29.28 (2C), 28.67, 26.45, 26.22 (2C), 25.05,22.66 (3C), 14.11 (3C), 11.78 (2C).

Example 12: 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl4,4-bis((2-ethylhexyl)oxy)butanoate

¹H NMR (400 MHz, CDCl₃) δ=4.80 (t, J=6.3 Hz, 1H), 4.46 (t, J=5.5 Hz,1H), 4.27-3.99 (m, 4H), 3.55-3.41 (m, 2H), 3.28 (dt, J=5.8, 8.9 Hz, 2H),2.56 (br. s., 4H), 2.39 (t, J=7.5 Hz, 2H), 2.00-1.77 (m, 6H), 1.75-1.53(m, 4H), 1.53-1.44 (m, 2H), 1.44-1.23 (m, 36H), 1.05 (d, J=6.8 Hz, 6H),0.98-0.76 (m, 15H). MS (M+1)=728.3, Rt=2.09 min (LC Method 4).

Example 13: 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl4,4-bis((2-propylpentyl)oxy)butanoate

¹H NMR (400 MHz, CDCl₃) δ=4.80 (t, J=6.1 Hz, 1H), 4.45 (t, J=5.6 Hz,1H), 4.25-4.06 (m, 4H), 3.47 (dd, J=5.8, 9.0 Hz, 2H), 3.27 (dd, J=5.8,9.3 Hz, 2H), 2.75-2.45 (m, 6H), 2.38 (t, J=7.7 Hz, 2H), 2.01-1.80 (m,6H), 1.72-1.47 (m, 4H), 1.43-1.18 (m, 36H), 1.18-0.98 (m, 6H), 0.97-0.81(m, 15H). 13C NMR (101 MHz, CDCl₃) δ=173.34, 154.89, 102.40, 75.58,69.00 (2C), 66.25, 60.63, 49.02, 46.81 (2C), 37.91 (2C), 34.19, 33.66(4C), 32.95, 31.90, 29.63 (3C), 29.55, 29.49, 29.44, 29.38, 29.34,28.63, 26.07, 25.04, 22.68, 19.96 (2C), 19.92 (2C), 14.47 (4C), 14.12,11.39 (2C).

Example 14: 3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl4,4-bis((2-propylpentyl)oxy)butanoate

¹H NMR (400 MHz, CDCl₃) δ=4.80 (t, J=6.1 Hz, 1H), 4.45 (t, J=5.5 Hz,1H), 4.27-4.06 (m, 4H), 3.47 (dd, J=5.8, 9.0 Hz, 2H), 3.27 (dd, J=5.8,9.0 Hz, 2H), 2.38 (t, J=7.5 Hz, 9H), 2.03-1.84 (m, 6H), 1.73-1.47 (m,4H), 1.38-1.09 (m, 39H), 0.96-0.78 (m, 15H). 13C NMR (101 MHz, CDCl₃)δ=173.37, 154.82, 102.39, 75.65, 69.01 (2C), 65.87 (br), 60.57, 53.27,51.30, 40.69 (br), 37.90 (2C), 34.19 (2C), 33.66 (4C), 32.94, 31.90,29.63 (2C), 29.55, 29.49, 29.44, 29.38, 29.34, 28.63, 25.87 (br), 25.05,22.68, 19.96 (2C), 19.92 (2C), 14.47 (4C), 14.12, 11.02.

Example 15: 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl4,4-bis((2-propylpentyl)oxy)butanoate

¹H NMR (400 MHz, CDCl₃) δ=4.91-4.69 (m, 1H), 4.45 (t, J=5.5 Hz, 1H),4.28-4.03 (m, 4H), 3.47 (dd, J=5.8, 9.0 Hz, 2H), 3.27 (dd, J=5.8, 9.3Hz, 2H), 2.52 (br. s., 2H), 2.44-2.26 (m, 8H), 2.02-1.85 (m, 6H),1.72-1.48 (m, 4H), 1.40-1.16 (m, 36H), 0.99-0.83 (m, 15H). ¹³C NMR (101MHz, CDCl₃) δ=173.36, 154.83, 102.39, 75.64, 69.03, 69.00, 65.90, 60.60,55.84, 44.90 (2C), 37.90 (2C), 34.19, 33.66 (4C), 32.94, 31.90, 29.65(2C), 29.62, 29.55, 29.49, 29.43, 29.38, 29.34, 28.63, 26.34, 25.04,22.68, 19.96 (2C), 19.92 (2C), 14.47 (4C), 14.12.

Synthesis of Example 16 Intermediate 16a: methyl 6,6-dimethoxyhexanoate

Methyl 6-oxohexanoate (11 g, 76 mmol) was taken into methanol (60 mL)and conc. sulfuric acid (244 uL, 4.58 mmol) was added. The reaction washeated to reflux overnight. The reaction was cooled to ambienttemperature and diluted with water. The mixture was extracted with ethylacetate. The combined organic extracts were dried over sodium sulfate,filtered, and concentrated under reduced pressure. The concentrate waspurified on silica gel with ethyl acetate/heptane as eluent to afford12.1 g of the desired compound. ¹H NMR (400 MHz, CDCl₃) δ=4.37 (t,J=5.77 Hz, 1H), 3.68 (s, 3H), 3.33 (s, 6H), 2.34 (t, J=7.53 Hz, 2H),1.57-1.73 (m, 4H), 1.34-1.46 (m, 2H) ppm.

Intermediate 16b: octyl 6,6-bis(octyloxy)hexanoate

Intermediate 16a (3.06 g, 16.09 mmol) was dissolved in 1-octanol (10.17ml, 64.3 mmol) and potassium bisulfate (0.110 g, 0.804 mmol) was addedThe mixture was heated to 70° C., and stirred for 4 h. The mixture wasdiluted with 40 mL of water, and extracted with EtOAc (2×50 mL). Theorganic layers were combined, washed with brine (100 mL), dried oversodium sulfate, then concentrated in vacuo. The crude mixture waspurified by chromatography on silica gel (0-10% EtOAc/heptane gradient)to provide 5.07 g of the title compound (65% yield). ¹H NMR (400 MHz,CDCl₃) δ=4.46 (t, J=5.69 Hz, 1H), 4.02-4.12 (m, 2H), 3.56 (dt, J=9.29,6.66 Hz, 2H), 3.40 (dt, J=9.32, 6.71 Hz, 2H), 2.31 (t, J=7.58 Hz, 2H),1.51-1.72 (m, 12H), 1.20-1.45 (m, 30H), 0.81-0.97 (m, 9H).

Intermediate 16c: 6,6-bis(octyloxy)hexanoic acid

To intermediate 16b (5.07 g, 10.46 mmol) was added water (34.9 ml), MeOH(34.9 ml), and NaOH (2.091 g, 52.3 mmol). The mixture was heated toreflux, and stirred for 2 h. The mixture was neutralized with 1N HCl,then extracted with EtOAc (2×50 mL). The organic layers were combined,washed with brine (100 mL), then dried over sodium sulfate, filtered,and concentrated. The crude material was used in the next step withoutfurther purification.

¹H NMR (400 MHz, DMSO-d₆) δ=4.38 (t, J=5.62 Hz, 1H), 3.42-3.51 (m, 2H),3.28-3.38 (m, 2H), 1.96 (t, J=7.34 Hz, 2H), 1.39-1.55 (m, 8H), 1.19 (m,23H), 0.80-0.93 (m, 6H).

Preparation of Final Compounds

The following examples (Examples 16-23) can be prepared using similarmethods to those employed for the synthesis of Example 1.

Example 16: 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate

¹H NMR (400 MHz, CDCl₃) δ=4.69-4.95 (m, 1H), 4.46 (t, J=5.65 Hz, 1H),4.06-4.27 (m, 4H), 3.56 (td, J=6.78, 9.29 Hz, 2H), 3.40 (td, J=6.78,9.29 Hz, 2H), 2.43-2.58 (m, 6H), 2.31 (t, J=7.65 Hz, 2H), 1.92 (q,J=6.27 Hz, 2H), 1.81 (td, J=6.87, 14.12 Hz, 2H), 1.49-1.73 (m, 12H),1.19 (br. s., 40H), 1.01 (t, J=7.15 Hz, 6H), 0.89 (t, J=6.78 Hz, 9H).¹³C NMR (101 MHz, CDCl₃) δ=173.54, 154.99, 102.81, 75.51, 66.57, 65.55(2C), 60.59, 49.03, 46.89 (2C), 34.20, 34.15, 33.13, 32.97, 31.91, 31.83(2C), 29.89 (2C), 29.64, 29.56 (2C), 29.49 (2C), 29.44 (3C), 29.35 (3C),29.28 (2C), 26.46, 26.26 (2C), 25.04, 24.74, 24.36, 22.66 (2C), 14.11(2C), 11.78 (2C).

Example 17: 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl6,6-bis(hexyloxy)hexanoate

¹H NMR (400 MHz, CDCl₃) δ=4.80 (t, J=6.15 Hz, 1H), 4.46 (t, J=5.77 Hz,1H), 4.04-4.27 (m, 4H), 3.56 (dt, J=9.29, 6.78 Hz, 2H), 3.40 (dt,J=9.29, 6.78 Hz, 2H), 2.55 (br. s., 4H), 2.31 (t, J=7.53 Hz, 2H), 1.92(q, J=6.53 Hz, 2H), 1.77-1.89 (m, 2H), 1.48-1.74 (m, 10H), 1.18-1.48 (m,36H), 1.04 (t, J=7.03 Hz, 6H), 0.89 (dq, J=6.68, 3.38 Hz, 9H). MS(M+1)=700.7, Rt=1.71 min (LC Method 4).

Example 18: 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl6,6-bis((2-ethylhexyl)oxy)hexanoate

¹H NMR (400 MHz, CDCl₃) δ=4.89-4.71 (m, 1H), 4.42 (t, J=5.6 Hz, 1H),4.28-4.06 (m, 4H), 3.46 (dt, J=6.0, 8.3 Hz, 2H), 3.37-3.17 (m, 2H), 2.55(br. s., 4H), 2.31 (t, J=7.5 Hz, 2H), 2.01-1.76 (m, 4H), 1.73-1.54 (m,6H), 1.54-1.43 (m, 2H), 1.43-1.23 (m, 40H), 1.04 (t, J=6.7 Hz, 6H),0.97-0.77 (m, 15H). MS (M+1)=756.5, Rt=2.23 min (LC Method 4).

Example 19: 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl8,8-bis(octyloxy)octanoate

¹H NMR (400 MHz, CDCl₃) δ=4.81 (t, J=6.15 Hz, 1H), 4.45 (t, J=5.77 Hz,1H), 4.05-4.26 (m, 4H), 3.56 (dt, J=9.22, 6.68 Hz, 2H), 3.40 (dt,J=9.22, 6.68 Hz, 2H), 2.57 (br. s., 6H), 2.29 (t, J=7.65 Hz, 2H),1.79-1.98 (m, 4H), 1.49-1.75 (m, 10H), 1.15-1.44 (m, 38H), 0.98-1.09 (m,6H), 0.75-0.98 (m, 9H). ¹³C NMR (101 MHz, CDCl₃) δ=173.71, 154.94,103.01, 75.58, 66.38, 65.44 (2C), 60.51, 49.01, 46.82 (2C), 34.19 (2C),33.39, 32.95, 31.90, 31.67 (2C), 29.85 (2C), 29.65 (2C), 29.62, 29.55,29.48, 29.43, 29.34, 29.13, 29.09, 26.14, 25.93 (2C), 25.04, 24.82,24.64, 22.68, 22.62 (2C), 14.12, 14.06 (2C), 11.44 (2C).

Example 20: 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl8,8-dibutoxyoctanoate

¹H NMR (400 MHz, CDCl₃) δ=4.46 (t, J=5.65 Hz, 1H) 4.81 (t, J=6.15 Hz,1H), 4.05-4.25 (m, 4H), 3.57 (dt, J=9.35, 6.62 Hz, 2H), 3.41 (dt,J=9.29, 6.65 Hz, 2H), 2.57 (br. s., 4H), 2.29 (t, J=7.53 Hz, 2H),1.72-1.97 (m, 4H), 1.48-1.72 (m, 12H), 1.19-1.48 (m, 30H), 1.06 (br. s.,6H), 0.78-0.99 (m, 9H). ¹³C NMR (101 MHz, CDCl₃) δ=173.72, 154.94,103.02, 75.59, 66.38, 65.12 (2C), 60.51, 49.03, 46.84 (2C), 34.19 (2C),33.38, 32.96, 31.97 (2C), 31.90, 29.65, 29.63 (2C), 29.56, 29.49, 29.44,29.35, 29.14, 29.09, 26.23, 25.04, 24.82, 24.63, 22.68, 19.44 (2C),14.13, 13.92 (2C), 11.48 (2C).

Example 21: 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl8,8-bis((2-propylpentyl)oxy)octanoate

¹H NMR (400 MHz, CDCl₃) δ=4.81 (t, J=6.3 Hz, 1H), 4.40 (t, J=5.6 Hz,1H), 4.26-4.06 (m, 4H), 3.45 (dd, J=5.6, 9.2 Hz, 2H), 3.26 (dd, J=5.9,9.2 Hz, 2H), 2.59 (br. s., 6H), 2.29 (t, J=7.5 Hz, 2H), 1.92 (q, J=6.3Hz, 4H), 1.72-1.49 (m, 10H), 1.38-1.16 (m, 40H), 1.07 (br. s., 6H),0.98-0.80 (m, 15H). ¹³C NMR (101 MHz, CDCl₃) δ=173.39, 154.59, 103.14,75.28, 68.10 (2C), 65.92, 60.16, 48.70, 46.50 (2C), 37.62 (2C), 33.87(2C), 33.42 (2C), 33.37 (2C), 32.99, 32.64, 31.58, 29.31 (3C), 29.24,29.16, 29.11, 29.03, 28.83, 28.81, 25.68, 24.72, 24.51, 24.38, 22.36,19.63 (4C), 14.16 (4C), 13.81, 11.01 (2C).

Example 22: 3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl8,8-bis((2-propylpentyl)oxy)octanoate

¹H NMR (400 MHz, CDCl₃) δ=4.80 (t, J=6.1 Hz, 1H), 4.40 (t, J=5.8 Hz,1H), 4.26-4.04 (m, 4H), 3.45 (dd, J=5.8, 9.3 Hz, 2H), 3.32-3.17 (m, 2H),2.51 (br. s., 4H), 2.36-2.20 (m, 5H), 1.92 (q, J=6.3 Hz, 4H), 1.73-1.46(m, 10H), 1.46-1.17 (m, 40H), 1.17-1.01 (m, 3H), 0.98-0.78 (m, 15H). ¹³CNMR (101 MHz, CDCl₃) δ=173.70, 154.90, 103.45 (2C), 75.61, 68.41 (2C),66.22, 60.47, 53.37, 51.37, 41.23, 37.94 (2C), 34.19 (2C), 33.73 (2C),33.69 (2C), 33.31, 32.95, 31.90, 29.62 (2C), 29.55, 29.48, 29.43, 29.34,29.15, 29.12, 26.25, 25.03, 24.83, 24.69, 22.68, 19.95 (4C), 14.48 (4C),14.12, 11.86.

Example 23: 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl8,8-bis((2-propylpentyl)oxy)octanoate

¹H NMR (400 MHz, CDCl₃) δ=4.80 (t, J=6.1 Hz, 1H), 4.40 (t, J=5.8 Hz,1H), 4.26-4.04 (m, 4H), 3.45 (dd, J=5.8, 9.3 Hz, 2H), 3.26 (dd, J=5.9,9.2 Hz, 2H), 2.53-2.36 (m, 2H), 2.36-2.20 (m, 8H), 1.91 (td, J=6.2, 12.2Hz, 4H), 1.71-1.48 (m, 10H), 1.44-1.16 (m, 40H), 0.98-0.79 (m, 15H). ¹³CNMR (101 MHz, CDCl₃) δ=173.71, 154.89, 103.45, 75.63, 68.41 (2C), 66.08,60.47, 55.90, 45.15 (2C), 37.94 (2C), 34.19 (2C), 33.73 (2C), 33.69(2C), 33.31, 32.94, 31.90, 29.62 (2C), 29.54 (2C), 29.48, 29.42, 29.34,29.14, 29.12, 26.61, 25.03, 24.83, 24.69, 22.68, 19.95 (4C), 14.48 (4C),14.12.

Synthesis of Example 24 Intermediate 24a: ethyl 3-octylundec-2-enoate

A solution of 9-heptadecanone (15 g, 59 mmol) andtriethylphosphonoacetate (13.2 g, 59 mmol) was stirred in THF (100 mL).To this mixture was added NaOEt (26.4 mL, 21% in EtOH, 70.7 mmol) andthe resulting solution was heated to reflux for 48 h. The reaction wasacidified with 1M HCl and then diluted with EtOAc. The organic layer wascollected and washed with saturated aqueous sodium bicarbonate. Theresulting organic material was dried over sodium sulfate and thevolatiles removed under reduced pressure to yield a crude material thatwas purified by silica gel chromatography using heptanes/EtOAc aseluent, providing 11.7 g of the desired product. ¹H NMR (400 MHz,CDCl₃): δ=5.62 (s, 1H), 4.01-4.26 (m, 2H), 2.49-2.68 (m, 2H), 2.13 (m,2H), 1.44 (dd, J=7.33, 4.80 Hz, 4H), 1.17-1.35 (m, 23H), 0.83-0.98 (m,6H).

Intermediate 24b: ethyl 3-octylundecanoate

Intermediate 24a (11.75 g, 36.2 mmol) was stirred in DCM (16.5 mL) andMeOH (165 mL). 10% Pd/C (3.85 g,) was added and the reaction flask wasfitted with a balloon filled with hydrogen. The reaction was stirred atroom temperature for 24 h. The reaction was degassed with nitrogen andfiltered through celite with a wash of DCM and MeOH. The filtrate wascollected and the volatiles removed under reduced pressure to provide10.6 g of the desired product, which was utilized without furtherpurification. ¹H NMR (400 MHz, CDCl₃) δ=(q, J=7.16 Hz, 2H), 2.39 (t,J=7.45 Hz, 2H), 2.22 (d, J=6.82 Hz, 2H), 1.84 (br. s., 1H), 1.56 (t,J=7.20 Hz, 2H), 1.19-1.36 (m, 27H), 0.81-0.95 (m, 6H).

Intermediate 24c: 3-octylundecanoic acid

Intermediate 24b (10.6 g, 32.5 mmol) was stirred with NaOH (9.74 mL, 10M, 97.4 mmol) in MeOH (100 mL) and DCM (10 mL). The reaction was heatedto reflux overnight. Aqueous HCl was added to neutralize the solution,the volatiles were removed under reduced pressure and the resultingmaterial was taken back up in DCM. The organics were washed with aqueoussaturated sodium bicarbonate and the resulting aqueous layer wasback-extracted with DCM. The combined organics were dried over magnesiumsulfate, filtered, and concentrated under reduced pressure. The crudematerial was purified by silica gel chromatography using heptanes/EtOAcas eluent. The resulting material was taken up in DCM and loaded onto anNH₂ functionalized column. The column was washed with DCM and thenDCM/MeOH. The product was eluted with acidic methanol and the eluentconcentrated under reduced pressure. The residue was taken up in DCM andwashed with saturated aqueous sodium bicarbonate, dried over magnesiumsulfate, filtered, and concentrated under reduced pressure to provide6.5 g of the desired product. ¹H NMR (400 MHz, CDCl₃) δ=2.28 (d, J=7.07Hz, 2H) 1.86 (br. s., 1H) 1.15-1.44 (m, 28H) 0.82-0.97 (m, 6H).

Example 24: 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl3-octylundecanoate

Example 24 can be prepared using similar methods to those employed forthe synthesis of Example 1

¹H NMR (400 MHz, CDCl₃) δ=4.71-4.88 (m, 1H) 4.01-4.31 (m, 4H) 2.71 (br.s., 2H) 2.50 (br. s., 6H) 2.17-2.28 (m, 2H) 2.06 (br. s., 2H) 1.88-1.99(m, 2H) 1.83 (br. s., 1H) 1.50-1.75 (m, 2H) 1.17-1.40 (m, 48H) 0.80-0.96(m, 9H). ¹³C NMR (101 MHz, CDCl₃) δ=173.27, 154.46, 75.52, 65.14, 59.94,55.37, 43.93 (2C), 38.90, 34.71, 33.89, 33.53 (2C), 32.72, 31.58 (3C),29.60, 29.34 (2C), 29.31 (2C), 29.28 (2C), 29.18, 29.14, 29.02 (2C),29.00 (2C), 26.21 (3C), 24.75, 22.35 (3C), 13.78 (3C).

Synthesis of Example 25 Intermediate 25a: 3-octylundec-2-enoic acid

Intermediate 25a can be synthesized from Intermediate 24a utilizingsimilar methods to those used for the synthesis of Intermediate 24c.

TLC (silica gel, 10% ethyl acetate in hexanes): R_(f)=0.18

Example 25: 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl3-octylundec-2-enoate

Example 25 can be prepared using similar methods to those employed forthe synthesis of Example 1

¹H NMR (400 MHz, CDCl₃) δ=5.20-5.41 (m, 1H) 4.81 (dt, J=13.45, 6.54 Hz,1H) 4.04-4.27 (m, 4H) 2.58 (m, 2H) 2.30-2.48 (m, 2H) 2.24 (s, 6H)1.99-2.16 (m, 4H) 1.82-1.98 (m, 4H) 1.50-1.74 (m, 2H) 1.43 (dd, J=14.15,6.57 Hz, 2H) 1.17-1.40 (m, 40H) 0.80-1.00 (m, 9H). MS (M+1)=652.5,Rt=1.44 min (LC Method 4).

Synthesis of Example 26 Intermediate 26a: methyl 3-hexylnon-2-enoate

To a suspension of sodium hydride (60% in paraffin oil, 14.16 g, 335mmol) in THF (500 mL), cooled in an ice-water bath, was slowly addedtrimethyl phosphonoacetate (50.74 g, 278.8 mmol) to control gasevolution. After the addition was finished, the reaction was stirred for2 h then tridecan-7-one (6.5 g, 32.8 mmol) was slowly added, and thereaction was warmed to ambient temperature. After 1 h additional, thereaction was heated to reflux. After 4 d, the reaction was cooled, and1N HCl (aq) was added to quench the reaction. The reaction was extractedwith ethyl acetate (3×200 mL). The combined organic extracts were driedover sodium sulfate, filtered, and concentrated under reduced pressure.The concentrate was purified on silica gel, using ethyl acetate/hexanesas eluent to provide 8.0 g of the desired product. TLC (silica gel, 10%ethyl acetate in hexanes): R_(f)=0.72.

Intermediate 26b: 3-hexylnon-2-en-1-ol

To a solution of Intermediate 26a (8.1 g, 31.9 mmol) in THF (100 mL),cooled in an ice-water bath, was added diisobutylaluminum hydride (25%in toluene, 54.4 mL, 95.6 mmol). After 30 minutes the reaction wasbrought to ambient temperature. After an additional 6 h, the reactionwas cooled in an ice-water bath and quenched with ice-cold water (50 mL)and 1N HCl (aq, 15 mL). The reaction was extracted with ethyl acetate(2×50 mL). The combined organic extracts were washed with water (2×60mL) and brine (60 mL). The organic extract was dried over sodiumsulfate, filtered, and concentrated under reduced pressure. Theconcentrate was purified on silica-gel, using ethyl acetate/hexanes aseluent to provide 6.8 g of the desired product. TLC (silica gel, 20%ethyl acetate in hexanes): R_(f)=0.29.

Intermediate 26c: 3-hexylnon-2-enal

To a stirred suspension of IBX (21.0 g, 75.12 mmol) in DMSO (30 mL),warmed to 30° C., was added Intermediate 26b in THF (100 mL). Thereaction was maintained at 25-30° C. for 2 h. The reaction was dilutedwith diethyl ether and filtered through celite with diethyl etherwashes. The filtrate was washed with water (2×200 mL) and brine (200mL). The organic extract was dried over sodium sulfate, filtered, andconcentrated under reduced pressure to provide 6.0 g of the desiredproduct, which was used without further purification. TLC (silica gel,10% ethyl acetate in hexanes): R_(f)=0.50.

Intermediate 26d: 7-hexyltrideca-4,6-dienoic acid

To a suspension of (3-carboxypropyl)triphenylphosphonium bromide (19.09g, 44.6 mmol) in THF (80 mL) and HMPA (5 mL), cooled in an ice-waterbath, was added NaHMDS (1.0M in THF, 111 mL, 111 mmol). Intermediate 26c(5.0 g, 22.3 mmol) in THF (20 mL), was slowly added, and the reactionwas warmed to 30° C. After 16 h, the reaction was diluted with 200 mLwater and acidified with 2N HCl (aq). The reaction was extracted withethyl acetate (3×100 mL). The combined organic extracts were dried oversodium sulfate, filtered, and concentrated under reduced pressure. Theconcentrate was purified on silica gel with ethyl acetate/hexanes aseluent to provide 4.0 g of the desired product. TLC (silica gel, 30%ethyl acetate in n-hexane): R_(f)=0.21.

Intermediate 26e: 7-hexyltridec-6-enoic acid

To a solution of Intermediate 26d (1.0 g) in methanol (60 mL) was added10% Pd/C (300 mg). The reaction was stirred at ambient temperature undera balloon of hydrogen gas for 14 h. The reaction mixture was filteredover celite and the residue rinsed with methanol. The filtrate wasconcentrated under reduced pressure. The concentrate was purified onsilica gel with ethyl acetate/hexanes as eluent to provide the desiredproduct. TLC (silica gel, 10% ethyl acetate in n-hexane): R_(f)=0.16

Example 26: 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl7-hexyltridec-6-enoate

Example 26 can be prepared using similar methods to those employed forthe synthesis of Example 1

¹H NMR (400 MHz, CDCl₃) δ=5.02-5.20 (m, 1H) 4.80 (t, J=5.18 Hz, 1H)4.00-4.30 (m, 4H) 2.74 (br. s., 2H) 2.53 (br. s., 6H) 2.30 (t, J=7.58Hz, 2H) 1.84-2.14 (m, 8H) 1.50-1.78 (m, 4H) 1.21-1.42 (m, 40H) 0.80-0.98(m, 9H). ¹³C NMR (101 MHz, CDCl₃) δ=173.41, 154.46, 139.95, 123.49,75.49, 65.05, 59.99, 55.37, 43.86 (2C), 36.62, 35.12, 33.90, 32.70,31.59 (2C), 31.50, 29.76, 29.33 (2C), 29.31 (3C), 29.24, 29.17, 29.12,29.02, 28.83, 28.15, 27.93, 26.99, 26.91, 24.74, 24.33, 22.36 (3C),13.78 (3C).

Synthesis of Example 27 Intermediate 27a:(E)-9-pentyltetradeca-6,8-dienoic acid

Intermediate 27a can be synthesized utilizing similar methods to thosein used to produce intermediate 26d.

TLC (silica gel, 10% ethyl acetate in n-hexane): R_(f)=0.31

Intermediate 27b: 9-pentyltetradecanoic acid

To a solution of Intermediate 27a (2.0 g, 6.80 mmol) in methanol (70 mL)was added 10% Pd/C (300 mg). The reaction was stirred at ambienttemperature under a balloon of hydrogen gas for 4 h. The reactionmixture was filtered over celite, and the residue washed with methanol.The filtrate was concentrated under reduced pressure. The concentratewas purified on silica gel with ethyl acetate/n-hexane as eluent toprovide the desired product.

TLC (silica gel, 10% ethyl acetate in n-hexane): R_(f)=0.26

Example 27: 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl9-pentyltetradecanoate

Example 27 can be prepared using similar methods to those employed forthe synthesis of Example 1

¹H NMR (400 MHz, CDCl₃) δ=4.81 (t, J=5.05 Hz, 1H) 4.01-4.30 (m, 4H)2.68-2.89 (m, 1H) 2.55 (br. s., 6H) 2.30 (t, J=7.58 Hz, 2H) 2.11 (br.s., 2H) 1.84-2.02 (m, 2H) 1.61 (dt, J=14.65, 7.33 Hz, 4H) 1.12-1.38 (m,48H) 0.77-0.99 (m, 9H). ¹³C NMR (101 MHz, CDCl₃) δ=173.81, 154.79,75.79, 65.46, 60.30, 55.71, 44.29 (2C), 37.42, 34.29, 34.21, 33.69,33.63 (s, 2C), 33.02, 32.38 (s, 2C), 31.91, 29.98, 29.65, 29.63 (s, 2C),29.56, 29.49, 29.43, 29.34 (s, 2C), 29.21, 26.68, 26.36 (s, 3C), 25.06,24.95, 22.71 (s, 2C), 22.68, 14.13 (s, 2C), 14.10.

Example 28: 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl9-pentyltetradec-8-enoate

Example 28 can be prepared using similar methods to those employed forthe synthesis of Example 26

¹H NMR (400 MHz, CDCl₃) δ=ppm 5.02-5.17 (m, 1H) 4.74-4.90 (m, 1H)4.05-4.27 (m, 4H) 2.38 (br. s., 2H) 2.16-2.34 (m, 8H) 1.80-2.08 (m, 10H)1.49-1.72 (m, 6H) 1.15-1.37 (m, 36H) 0.81-0.97 (m, 9H). MS (M+1)=652.3,Rt=1.41 min (LC Method 4).

Synthesis of Example 29 Intermediate 29a: 3-heptyldec-2-enal

Intermediate 29a can be prepared using similar methods to those employedfor the preparation of Intermediate 26c.

TLC (silica gel, 10% ethyl acetate in hexanes): R_(f)=0.63.

Intermediate 29b: methyl 5-heptyldodeca-2,4-dienoate

To a suspension of sodium hydride (55% in paraffin oil, 3.5 g, 74.3mmol) in THF (70 mL), cooled in an ice-water bath, was slowly addedtrimethylphosphonoacetate (9.6 mL, 59.5 mmol). After 10 min,Intermediate 29a (7.5 g, 29.7 mmol) in THF (10 mL) was added, and thereaction was allowed to warm to ambient temperature. After an additional2 h, the reaction was quenched by slow addition of ice-cold water (20mL). The reaction was extracted with ethyl acetate (2×100 mL). Theorganic extracts were washed with water and brine. The organics weredried over sodium sulfate, filtered, and concentrated under reducedpressure to provide 8.0 g of the desired product, which was used withoutfurther purification. TLC (silica gel, 10% ethyl acetate in hexanes):R_(f)=0.75.

Intermediate 29c: methyl 5-heptyldodecanoate

To a solution of Intermediate 29b (8.0 g, 25.95 mmol) in methanol (350mL) was added 10% Pd/C (1.0 g). The reaction was carried out under aatmosphere of hydrogen delivered by a balloon. After 14 h, the reactionwas filtered through celite with methanol washes. The filtrate wasconcentrated under reduced pressure to provide 7.7 g of the desiredproduct, which was used without further purification. TLC (silica gel,5% methanol in dichloromethane): R_(f)=0.63.

Intermediate 29d: 5-heptyldodecanoic acid

To a mixture of 5N aq sodium hydroxide (125 mL) and methanol (350 mL)was added Intermediate 29c (7.7 g, 24.7 mmol), and the reaction washeated to reflux. After 16 h, the reaction was cooled in an ice-waterbath and quenched by addition of concentrated aqueous HCl until acidic.The mixture was extracted with ethyl acetate (2×250 mL). The organicextracts were dried over sodium sulfate, filtered, and concentratedunder reduced pressure. The concentrate was purified on silica gel withethyl acetate/hexanes as eluent to provide 7.0 g of the desired product.Rf=0.82, 50% EtOAc/heptane

The following examples (Examples 29-31) can be prepared using similarmethods to those employed for the synthesis of Example 1.

Example 29: 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl5-heptyldodecanoate

¹H NMR (400 MHz, CDCl₃) δ=4.69-4.88 (m, 1H) 4.03-4.30 (m, 4H) 2.64 (br.s., 2H) 2.45 (br. s., 6H) 2.28 (t, J=7.58 Hz, 2H) 1.97-2.12 (m, 2H)1.85-1.97 (m, 2H) 1.51-1.73 (m, 3H) 1.14-1.38 (m, 48H) 0.82-0.96 (m,9H). ¹³C NMR (101 MHz, CDCl₃) δ=173.79, 154.82, 75.79, 65.59, 60.37,55.75, 44.50 (2C), 37.23, 34.67, 34.20, 33.50 (2C), 33.21, 33.02, 31.92(2C), 31.90, 30.07 (2C), 29.65, 29.63 (2C), 29.56, 29.48, 29.43, 29.36(2C), 29.33, 26.65 (2C), 25.95, 25.05, 22.67 (3C), 22.18, 14.10 (3C).

Example 30: 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)tridecyl5-heptyldodecanoate

¹H NMR (400 MHz, CDCl₃) δ=0.89 (t, J=6.65 Hz, 9H) 1.15-1.42 (m, 42H)1.49-1.77 (m, 5H) 1.77-1.99 (m, 4H) 2.18-2.37 (m, 8H) 2.43 (br. s., 2H)4.02-4.29 (m, 4H) 4.81 (t, J=6.27 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃)δ=173.81, 154.90, 75.66, 66.14, 60.51, 55.93, 45.23 (2C), 37.19, 34.65,34.19, 33.44 (2C), 33.14, 32.96, 31.93 (2C), 31.89, 30.07 (2C), 29.58,29.56, 29.49, 29.44, 29.38 (2C), 29.32, 26.70, 26.63 (2C), 25.04, 22.69(3C), 22.13, 14.13 (3C).

Example 31: 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)undecyl5-heptyldodecanoate

¹H NMR (400 MHz, CDCl₃) δ=0.76-0.97 (m, 9H) 1.16-1.46 (m, 40H) 1.46-1.75(m, 5H) 1.92 (dt, J=12.61, 6.37 Hz, 4H) 2.17-2.39 (m, 6H) 2.39-2.56 (m,2H) 4.03-4.33 (m, 4H) 4.81 (t, J=6.15 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃)δ=173.82, 154.88, 75.67, 66.07, 60.49, 55.82, 45.02 (2C), 37.19, 34.64,34.17, 33.43 (2C), 33.14, 32.95, 31.92 (2C), 31.82, 30.07 (2C), 29.42(2C), 29.38 (2C), 29.21, 26.62 (2C), 26.50, 25.03, 22.68 (2C), 22.64,22.12, 14.13 (2C), 14.10.

Synthesis of Example 32 Intermediate 32a:4-((1,3-bis(octanoyloxy)propan-2-yl)oxy)-4-oxobutanoic acid

In a 30 mL microwave vial equipped with a stirbar, 1,3-Dicaprylin (2 g,5.81 mmol) and succinic anhydride (0.581 g, 5.81 mmol) are dissolved inToluene (Volume: 10 ml). DMAP (0.284 g, 2.322 mmol) is added, andmixture microwaved at 140 C for 40 min. The crude mixture was evaporatedunder reduced pressure to obtain a crude oil. Purification by silica gelcolumn chromatography (80 g column, 0-10% EtOAc/Heptane) provided thetitle compound as a colorless oil (0.900 g, 70%). ¹H NMR (400 MHz,CDCl₃) δ=5.35-5.22 (m, 1H), 4.39-4.25 (m, 2H), 4.24-4.09 (m, 2H),2.76-2.60 (m, 4H), 2.39-2.26 (m, 4H), 1.70-1.53 (m, 4H), 1.39-1.19 (m,16H), 0.97-0.82 (m, 6H). ¹³C NMR (101 MHz, CDCl₃) δ=177.01, 173.38 (s,2C), 171.22, 69.57, 61.12 (s, 2C), 33.97 (s, 2C), 31.63 (s, 2C), 29.02(s, 2C), 28.89 (s, 2C), 28.74, 28.60, 24.80 (s, 2C), 22.58 (s, 2C),14.29 (s, 2C).

The following examples (Examples 32 and 33) can be prepared usingsimilar methods to those employed for the synthesis of Example 1.

Example 32: 1,3-bis(octanoyloxy)propan-2-yl(3-(((2-(dimethylamino)ethoxy)carbonyl)oxy)pentadecyl) succinate

¹H NMR (400 MHz, CDCl₃) δ=5.31-5.22 (m, 1H), 4.81 (s, 1H), 4.41-4.23 (m,4H), 4.23-4.09 (m, 4H), 2.79-2.58 (m, 6H), 2.46-2.25 (m, 10H), 2.01-1.86(m, 2H), 1.74-1.51 (m, 7H), 1.42-1.18 (m, 35H), 0.97-0.82 (m, 9H). MS(M+1)=787.0, Rt=1.60 min (LC Method 5).

Example 33: 1,3-bis(octanoyloxy)propan-2-yl(3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl) succinate

¹H NMR (400 MHz, CDCl₃) δ=5.33-5.22 (m, 1H), 4.80 (br. s., 1H),4.36-4.25 (m, 2H), 4.24-4.08 (m, 6H), 2.70-2.51 (m, 6H), 2.45-2.27 (m,10H), 2.01 (s, 2H), 2.00-1.86 (m, 4H), 1.70-1.51 (m, 6H), 1.39-1.18 (m,34H), 0.95-0.82 (m, 9H). MS (M+1)=801.7, Rt=1.27 min (LC Method 4).

Synthesis of Example 34 Intermediate 34a: 10-(octyloxy)-10-oxodecanoicacid

To a solution of sebacic acid (5.0 g, 24.7 mmol) in dichloromethane (40mL) was added EDC.HCl (7.2 g, 37.1 mmol) and DMAP (3.0 g, 24.7 mmol).The reaction was stirred at ambient temperature for 1 h, then 1-octanol(2.9 g, 22.2 mmol) was added. The reaction was stirred for an additional72 h. The reaction was diluted with water and the aqueous was extractedwith dichloromethane (2×30 mL). The combined dichloromethane extractswere washed with brine, dried over sodium sulfate, and filtered. Thefiltrate was concentrated under reduced pressure, and the resultingcrude material was purified on silica gel with ethyl acetate and hexaneas eluent to provide the desired product. TLC (silica gel, 40% ethylacetate in hexanes): R_(f)=0.42

The following examples (Examples 34-38) can be prepared using similarmethods to those employed for the synthesis of Example 1.

Example 34: 1-(3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl)10-octyl decanedioate

¹H NMR (400 MHz, CDCl₃) δ=4.69-4.94 (m, 1H) 4.19 (t, J=6.53 Hz, 2H) 4.13(t, J=6.53 Hz, 2H) 4.06 (t, J=6.78 Hz, 2H) 2.42 (t, J=6.40 Hz, 2H)2.21-2.37 (m, 10H) 1.76-1.98 (m, 4H) 1.50-1.69 (m, 8H) 1.18-1.41 (m,38H) 0.78-0.98 (m, 6H). MS (M+1)=670.5, Rt=1.11 min (LC Method 4).

Example 35: 1-(3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl)10-octyl decanedioate

¹H NMR (400 MHz, CDCl₃) δ=4.81 (s, 1H), 4.24-4.09 (m, 4H), 4.06 (t,J=6.8 Hz, 2H), 2.56 (br. s., 6H), 2.29 (t, J=7.5 Hz, 4H), 1.99-1.78 (m,4H), 1.61 (d, J=6.3 Hz, 8H), 1.41-1.18 (m, 38H), 1.05 (t, J=6.8 Hz, 6H),0.88 (t, J=6.8 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 174.0, 173.7, 154.9,75.6, 66.4, 64.4, 60.5, 49.0, 46.8 (2C), 34.3, 34.2 (2C), 33.0, 31.9,31.8, 29.7, 29.6 (2C), 29.5, 29.4, 29.3, 29.2 (3C), 29.1 (4C), 28.6,26.1, 25.9, 25.0 (2C), 24.8, 22.7, 22.6, 14.1 (2C), 11.4 (2C).

Example 36:1-(3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl) 10-octyldecanedioate

¹H NMR (400 MHz, CDCl₃) δ=4.81 (s, 1H), 4.10-4.25 (m, 4H), 4.06 (t,J=6.78 Hz, 2H), 2.50 (br. s., 4H), 2.19-2.38 (m, 7H), 1.83-1.99 (m, 4H),1.61 (d, J=6.27 Hz, 8H), 1.18-1.42 (m, 38H), 1.09 (t, J=7.03 Hz, 3H),0.89 (t, J=6.78 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ=174.0, 173.7, 154.9,75.6, 66.3, 64.4, 60.5, 53.3, 51.3, 41.2, 34.3, 34.2 (2C), 32.9, 31.9,31.8, 29.7 (3C), 29.6, 29.5, 29.4, 29.3, 29.2 (2C), 29.1 (4C), 28.6,26.3, 25.9, 25.1, 25.0, 24.8, 22.7, 22.6, 14.1 (2C), 11.9.

Example 37: 1-(3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl)10-(2-ethylhexyl) decanedioate

¹H NMR (400 MHz, CDCl₃) δ=4.90-4.68 (m, 1H), 4.28-4.08 (m, 4H), 3.98(dd, J=1.9, 5.9 Hz, 2H), 2.53 (d, J=6.5 Hz, 6H), 2.29 (dt, J=3.5, 7.5Hz, 4H), 1.92 (q, J=6.5 Hz, 2H), 1.83 (td, J=6.5, 13.6 Hz, 2H),1.74-1.48 (m, 7H), 1.43-1.17 (m, 36H), 1.03 (t, J=7.0 Hz, 6H), 0.96-0.82(m, 9H). ¹³C NMR (101 MHz, CDCl₃) δ=174.08, 173.73, 154.97, 75.55,66.61, 66.49, 60.54, 49.03, 46.87 (2C), 38.68, 34.39, 34.20 (2C), 32.96,31.90, 30.37, 29.65, 29.62 (2C), 29.55, 29.48, 29.43, 29.34, 29.10 (3C),28.89, 26.35 (2C), 25.04, 24.99, 24.85, 23.74, 22.96, 22.68, 14.12,14.05, 11.66 (2C), 10.98.

Example 38:1-(3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl)10-(2-ethylhexyl) decanedioate

¹H NMR (400 MHz, CDCl₃) δ=4.81 (t, J=6.1 Hz, 1H), 4.26-4.08 (m, 4H),3.98 (dd, J=1.9, 5.9 Hz, 2H), 2.45 (d, J=6.8 Hz, 4H), 2.30 (dt, J=3.4,7.5 Hz, 4H), 2.24 (s, 3H), 1.97-1.81 (m, 4H), 1.72-1.50 (m, 7H),1.43-1.20 (m, 36H), 1.07 (t, J=7.2 Hz, 3H), 0.96-0.82 (m, 9H). ¹³C NMR(101 MHz, CDCl₃) δ=174.08, 173.73, 154.97, 75.55, 66.61, 66.49, 60.54,49.0, 46.87 (2C), 38.68, 34.39, 34.20 (2C), 32.96, 31.90, 30.37, 29.65,29.62 (2C), 29.55, 29.48, 29.43, 29.34, 29.10 (3C), 28.89, 26.35 (2C),25.04, 24.99, 24.85, 23.74, 22.96, 22.68, 14.12, 14.05, 11.66 (2C),10.98.

Synthesis of Example 39 Intermediate 39a: 10-(octanoyloxy)decanoic acid

To a solution of octanoic acid (766 mg, 5.32 mmol) in dichloromethane(25 mL) was added EDC.HCl (1.02 g, 5.32 mmol) and DIPEA (0.95 mL, 5.32mmol). The reaction was stirred for 1 h at ambient temperature, then10-hydroxydecanoic acid (500 mg, 2.66 mmol) and DMAP (162 mg, 1.33 mmol)were added. The reaction was stirred for an additional 24 h. Thereaction was diluted with water, and the aqueous layer was extractedwith dichloromethane (2×30 mL). the combined dichloromethane extractswere washed with brine, dried over sodium sulfate and filtered. Thefiltrate was concentrated under reduced pressure and the crude materialwas purified on silica gel with methanol/dichloromethane as eluent toprovide the desired product. TLC (silica gel, 10% methanol indichloromethane): R_(f)=0.69

The following examples (Examples 39-42) can be prepared using similarmethods to those employed for the synthesis of Example 1.

Example 39: 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl10-(octanoyloxy)decanoate

¹H NMR (400 MHz, CDCl₃) δ=4.72-4.91 (m, 1H) 4.13-4.35 (m, 4H) 3.97-4.13(m, 4H) 2.85 (br. s., 2H) 2.62 (br. s., 6H) 2.30 (t, J=7.45 Hz, 4H) 2.14(br. s., 2H) 1.81-2.04 (m, 2H) 1.50-1.73 (m, 8H) 1.15-1.42 (m, 36H)0.79-0.98 (m, 6H). MS (M+1)=670.8, Rt=1.19 min (LC Method 4).

Example 40:8-dodecyl-2-methyl-6,12-dioxo-5,7,11-trioxa-2-azanonadecan-19-yldecanoate

¹H NMR (400 MHz, CDCl₃) δ=4.88-4.74 (m, 1H), 4.25 (t, J=5.4 Hz, 2H),4.21-4.09 (m, 2H), 4.05 (t, J=6.7 Hz, 2H), 2.66 (br. s., 2H), 2.41-2.24(m, 9H), 1.92 (q, J=6.4 Hz, 2H), 1.73-1.51 (m, 9H), 1.48-1.18 (m, 38H),0.96-0.81 (m, 6H). MS (M+1)=657.9, Rt=1.76 min (LC Method 4).

Example 41: 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl10-(octanoyloxy)decanoate

¹H NMR (400 MHz, CDCl₃) δ=0.80-0.94 (m, 6H) 1.03 (t, J=6.53 Hz, 6H)1.18-1.41 (m, 38H) 1.52-1.72 (m, 8H) 1.84 (br. s., 2H) 1.92 (q, J=6.36Hz, 2H) 2.29 (t, J=7.53 Hz, 4H) 2.54 (br. s., 6H) 4.05 (t, J=6.78 Hz,2H) 4.10-4.28 (m, 4H) 4.81 (t, J=6.27 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃)□=174.04, 173.75, 154.96, 75.56, 66.46, 64.35, 60.53, 49.03, 46.86 (2C),34.38, 34.22, 34.19, 32.96, 31.90, 31.65, 29.65, 29.63 (2C), 29.56,29.48, 29.43, 29.34, 29.33, 29.19 (2C), 29.11, 29.10, 28.92, 28.62,26.51-26.06 (m, 1C), 25.90, 25.04, 25.00, 24.86, 22.68, 22.59, 14.12,14.07, 11.59 (2C).

Example 42: 3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl10-(octanoyloxy)decanoate

¹H NMR (400 MHz, CDCl₃) δ=4.93-4.67 (m, 1H), 4.25-4.09 (m, 4H), 4.05 (t,J=6.7 Hz, 2H), 2.42 (q, J=7.4 Hz, 4H), 2.29 (t, J=7.5 Hz, 4H), 2.21 (s,3H), 2.00-1.79 (m, 4H), 1.67-1.50 (m, 8H), 1.41-1.15 (m, 38H), 1.05 (t,J=7.2 Hz, 3H), 0.88 (t, J=6.7 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃)δ=174.03, 173.74, 154.96, 75.57, 66.47, 64.35, 60.54, 53.46, 51.42,41.56, 34.38, 34.21, 34.18, 32.95, 31.90, 31.65, 29.62 (3C), 29.55,29.48, 29.43, 29.33 (2C), 29.19 (2C), 29.10 (2C), 28.92, 28.61, 26.63,25.90, 25.03, 25.00, 24.86, 22.68, 22.59, 14.12, 14.07, 12.28.

The following examples (Examples 43-46) can be prepared using similarmethods to those employed for the synthesis of Example 1.

Example 43:(9Z,12Z)-3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyloctadeca-9,12-dienoate

¹H NMR (400 MHz, CDCl₃) δ=5.23-5.48 (m, 4H) 4.81 (t, J=5.31 Hz, 1H)4.01-4.29 (m, 4H) 2.71-2.85 (m, 2H) 2.63 (br. s., 2H) 2.44 (br. s., 6H)2.30 (t, J=7.58 Hz, 2H) 2.06 (q, J=7.07 Hz, 6H) 1.84-1.97 (m, 2H)1.49-1.71 (m, 4H) 1.34-1.42 (m, 4H) 1.18-1.34 (m, 30H) 0.79-0.98 (m,6H). MS (M+1)=636.5, Rt=1.12 min (LC Method 6).

Example 44:(9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyloctadeca-9,12-dienoate

¹H NMR (400 MHz, CDCl₃) δ=0.79-0.98 (m, 6H) 1.04 (t, J=7.15 Hz, 6H)1.16-1.45 (m, 34H) 1.51-1.72 (m, 5H) 1.76-1.99 (m, 5H) 2.05 (q, J=6.78Hz, 4H) 2.30 (t, J=7.65 Hz, 2H) 2.47-2.64 (m, 4H) 2.78 (t, J=6.53 Hz,2H) 4.05-4.28 (m, 4H) 4.81 (t, J=6.15 Hz, 1H) 5.24-5.53 (m, 4H). MS(M+1)=665.6, Rt=1.40 min (LC Method 7).

Example 45:(9Z,12Z)-3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyloctadeca-9,12-dienoate

¹H NMR (400 MHz, CDCl₃) δ=5.29-5.46 (m, 4H), 4.83 (quin, J=6.20 Hz, 1H),4.20 (t, J=6.53 Hz, 2H), 4.15 (t, J=6.50 Hz, 2H), 2.79 (t, J=6.53 Hz,2H), 2.52 (br. s., 4H), 2.23-2.37 (m, 5H), 2.07 (q, J=6.78 Hz, 4H), 1.94(q, J=6.50 Hz, 4H), 1.53-1.78 (m, 4H), 1.23-1.43 (m, 34H), 1.06-1.18 (m,3H), 0.86-0.96 (m, 6H). MS (M+1)=650.6, Rt=1.92 min (LC Method 7).

Example 46:(9Z,12Z)-3-(((2-(dimethylamino)ethoxy)carbonyl)oxy)pentadecyloctadeca-9,12-dienoate

¹H NMR (400 MHz, CDCl₃) δ=5.45-5.28 (m, 4H), 4.81 (t, J=6.1 Hz, 1H),4.24 (t, J=5.6 Hz, 2H), 4.19-4.07 (m, 2H), 2.78 (t, J=6.7 Hz, 2H), 2.64(br. s., 2H), 2.38-2.25 (m, 8H), 2.05 (q, J=6.9 Hz, 4H), 1.92 (q, J=6.4Hz, 2H), 1.73-1.51 (m, 5H), 1.43-1.20 (m, 33H), 0.89 (dt, J=4.0, 6.8 Hz,6H). MS (M+1)=623.3, Rt=1.70 min (LC Method 7).

Synthesis of Example 47:1-((9Z,12Z)-octadeca-9,12-dienoyloxy)pentadecan-3-yl1,4-dimethylpiperidine-4-carboxylate Intermediate 47a:1-((tert-butyldimethylsilyl)oxy)pentadecan-3-yl1,4-dimethylpiperidine-4-carboxylate

In a round-bottom flask equipped with a stir bar,1,4-dimethylpiperidine-4-carboxylic acid hydrochloride (0.612 g, 3.07mmol) is dissolved in DCM (Volume: 20 ml). DIPEA (1.461 ml, 8.36 mmol)is added, followed by DMAP (0.136 g, 1.115 mmol), Intermediate 1g (1 g,2.79 mmol), and finally, EDC.HCl (0.695 g, 3.62 mmol). Mixture wasstirred at rt overnight. The volitiles were evaporated under reducedpressure. The crude mixture was purified by silica gel columnchromatography (80 g column, 0-30% EtOAc/Heptane, then 0-5% MeOH/DCM) toprovide the title compound as a colorless oil (0.373 g, 27%). ¹H NMR(400 MHz, CDCl₃) δ=5.00 (d, J=6.3 Hz, 1H), 3.73-3.53 (m, 2H), 2.98-2.54(m, 2H), 2.28 (s, 3H), 2.15 (m, 3H), 1.78 (q, J=6.8 Hz, 2H), 1.56 (d,J=6.8 Hz, 4H), 1.25 (s, 21H), 1.19 (s, 3H), 0.99-0.82 (m, 12H),0.16-0.00 (m, 6H).

Intermediate 47b: 1-hydroxypentadecan-3-yl1,4-dimethylpiperidine-4-carboxylate

In a 250 ml round-bottom flask equipped with a stir bar, Intermediate47a (373 mg, 0.749 mmol) is dissolved in MeOH (Volume: 10 ml) at rt. CAN(1068 mg, 1.948 mmol) is added, and mixture is stirred at rt for 2 hrs.Sat. sodium bicarbonate solution and DCM are added to the mixture in aseparatory funnel. The organics are then washed with bicarb, extractedin DCM, dried over MgSO4, filtered and concentrated under pressure togive crude product mixture. Purification by silica gel columnchromatography (80 g column, 0-10% MeOH/DCM) afforded the title compoundas a colorless oil in quantitative yield (287 mg, 100%). Rf=0.20, 5%MeOH.DCM

Example 47: 1-((9Z,12Z)-octadeca-9,12-dienoyloxy)pentadecan-3-yl1,4-dimethylpiperidine-4-carboxylate

In a 50 ml round-bottom flask equipped with a stirbar, Linoleic acid(252 mg, 0.898 mmol) and Intermediate 47b (287 mg, 0.748 mmol) aredissolved in DCM (Volume: 10 ml). DIPEA (0.523 ml, 2.99 mmol) is added,followed by DMAP (36.6 mg, 0.299 mmol) and finally, EDC.HCl (229 mg,1.197 mmol). Mixture is stirred at rt overnight. The solvent wasevaporated under pressure. Purification by silica gel chromatography (40g column, 0-60% EtOAc/heptanes) afforded the title compound as acolorless oil (270 mg, 53%). ¹H NMR (400 MHz, CDCl₃) δ=5.46-5.27 (m,4H), 5.01 (br. s., 1H), 4.17-4.00 (m, 2H), 2.77 (t, J=6.5 Hz, 2H),2.73-2.56 (m, 2H), 2.37-2.24 (m, 6H), 2.15 (d, J=12.8 Hz, 4H), 2.05 (q,J=6.8 Hz, 5H), 1.98-1.81 (m, 3H), 1.69-1.48 (m, 7H), 1.44-1.22 (m, 30H),1.20 (s, 3H), 0.89 (dt, J=4.3, 6.8 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃)δ=176.3, 173.8, 130.2, 130.0, 128.1, 127.9, 70.8, 60.5 (2C), 53.0 (2C),46.1, 41.0, 34.6, 34.2, 34.1, 33.1, 31.9, 31.5, 29.7, 29.6 (3C), 29.5(2C), 29.4, 29.3 (3C), 29.2, 29.1, 27.2 (2C), 25.6, 25.1, 24.9, 22.7,22.6 (2C), 14.1 (2C).

Synthesis of Example 48 Intermediate 48a:1-((tert-butyldimethylsilyl)oxy)tetradecan-2-ol

A suspension of tetradecane-1,2-diol (10 g, 43.4 mmol), imidazole (2.95g, 43.4 mmol) and TBSCl (7.20 g, 477 mmol) in THF was stirred for 15 hat rt, then poured into water (300 mL) and extracted with EtOAc. Theorganic extract was washed with brine and dried over MgSO4, filtered,and concentrated under reduced pressure. The residue was purified onsilica gel with EtOAc/heptane as eluent to afford 11.96 g of the desiredproduct. ¹H NMR (400 MHz, CDCl₃) δ=3.59-3.70 (m, 2H), 3.31-3.44 (m, 1H),2.44 (br. s., 1H), 1.35-1.51 (m, 2H), 1.26 (s, 20H), 0.81-0.98 (m, 12H),0.08 (s, 6H).

Intermediate 48b: 1-((tert-butyldimethylsilyl)oxy)tetradecan-2-yl(3-(diethylamino)propyl) carbonate

Intermediate 48b can be prepared using similar methods to those employedfor the synthesis of Intermediate 1j. ¹H NMR (400 MHz, CDCl₃)δ=4.66-4.83 (m, 1H), 4.18 (t, J=6.53 Hz, 2H), 3.66 (d, J=5.27 Hz, 2H),2.57 (br. s., 6H), 1.86 (d, J=5.77 Hz, 2H), 1.48-1.78 (m, 2H), 1.26 (s,20H), 1.05 (t, J=6.65 Hz, 6H), 0.81-0.97 (m, 12H), 0.05 (d, J=1.76 Hz,6H).

Intermediate 48c: 3-(diethylamino)propyl (1-hydroxytetradecan-2-yl)carbonate

In a plastic tube, Intermediate 48b (3.27 g, 6.52 mmol) was dissolved in10 mL THF and cooled in an ice bath. HF.Py (3.43 mL, 195 mmol) was addeddropwise. The mixture was then warmed to ambient temperature and stirredfor one hour. Sat. NaHCO₃ was added and the aqueous phase was extractedwith EtOAc three times. The organic phases were combined and dried overMgSO4, filtered, and concentrated under reduced pressure to provide 2.5g of the desired product, which was used without further purification.Rf=0.25, 15% MeOH in DCM

Example 48: 2-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecyl4,4-bis((2-ethylhexyl)oxy)butanoate

Example 48 can be prepared using similar methods to those employed forthe synthesis of Example 12. ¹H NMR (400 MHz, CDCl₃) δ=4.83-4.96 (m,1H), 4.45 (t, J=5.40 Hz, 1H), 4.30 (dd, J=11.92, 2.64 Hz, 1H), 4.14-4.26(m, 2H), 4.05 (dd, J=12.05, 6.53 Hz, 1H), 3.42-3.54 (m, 2H), 3.28 (td,J=9.03, 6.02 Hz, 2H), 2.59 (br. s., 6H), 2.41 (t, J=7.65 Hz, 2H),1.79-2.01 (m, 3H), 1.52-1.74 (m, 3H), 1.19-1.52 (m, 38H), 1.07 (br. s.,6H), 0.77-0.96 (m, 15H). MS (M+1)=715.0, Rt=1.96 min (LC Method 11).

Synthesis of Example 49 Intermediate 49a:(12Z,15Z)-1-((tert-butyldimethylsilyl)oxy)henicosa-12,15-dien-3-ol

Magnesium turnings (0.516 mg, 21.2 mmol) were weighed into a pre-dried100 mL flask. The flask was charged with nitrogen, sealed with a septumpierced with a 16G needle outlet and put in an oven at 120° C. for 2hours. The flask was removed from the oven, the needle was removed, andthe flask was allowed to cool to ambient temperature. To the flask, 40mL of anhydrous THF and a catalytic amount of iodine was added, followedby the addition of linoelyl bromide (5.25 g, 16.0 mmol). The flask wasconnected with a condenser and the reaction was refluxed under N₂ untilmost of magnesium was consumed (about 1 hr), then cooled to roomtemperature.

To a solution of 3-(tert-butyldimethylsilyloxy) propanal (Intermediate1g, 2.5 g, 13.3 mmol) in 50 ml THF, cooled in an ice-water bath, theabove prepared Grinard reagent was added dropwise. The reaction stirredfor 30 min. The reaction was quenched with Sat. NaHCO₃, and extractedwith ethyl acetate. The combined organic extracts were dried,concentrated under reduced pressure, and purified on silica gel with 10%ethylacetate/heptane as eluent to afford 2.7 g of the desired product.1H NMR (400 MHz, CDCl₃) δ=5.17-5.37 (m, 4H), 3.78-3.87 (m, 1H),3.67-3.78 (m, 2H), 2.68 (t, J=4.0 Hz, 2H), 1.89-2.05 (m, 4H), 1.53-1.64(m, 2H), 1.12-1.44 (m, 18H), 0.77-0.85 (m, 14H), 0.00 (s, 6H).

Intermediate 49b: (12Z,15Z)-henicosa-12,15-diene-1,3-diol

To a solution of Intermediate 49a (1.1 g, 2.5 mmol) in 15 ml THF at roomtemperature, TBAF (3.01 ml 1.0M THF solution, 3.01 mmol) was added. Thereaction was stirred for 1 hr. The reaction was extracted between brineand ethyl acetate. The combined organics were dried over sodium sulfate,concentrated under reduced pressure, and purified on silica gel with 50%ethylacetate/heptane as eluent to afford the desired product (0.78 g,96%). ¹H NMR (CDCl₃) δ=5.24-5.56 (m, 4H), 3.82-4.00 (m, 3H), 2.70-2.87(m, 2H), 2.19 (br. s., 2H), 2.07 (q, J=6.7 Hz, 4H), 1.64-1.82 (m, 2H),1.20-1.54 (m, 20H), 0.89-0.97 (m, 3H)

Intermediate 49c

To a solution of Intermediate 49b (390 mg, 1.20 mmol) in DCM (10 mL),linoleic acid (404 mg, 1.44 mmol) was added, followed by DIPEA (0.210mL, 1.20 mmol), DMAP (58.7 mg, 0.48 mmol), and EDC.HCl (323 mg, 1.68mmol). The reaction stirred at room temperature for 90 min. The reactionwas extracted between brine and ethyl acetate. The combined organicswere dried over sodium sulfate and filtered. The filtrate wasconcentrated under reduced pressure and the residue was purified onsilica gel with 15% ethyl acetate/heptane as eluent to provide the titlecompound. ¹H NMR (400 MHz, CDCl₃) δ=5.30-5.45 (m, 8H), 4.39 (ddd,J=11.23, 8.60, 5.02 Hz, 1H), 4.15 (dt, J=11.17, 5.71 Hz, 1H), 3.61-3.73(m, 1H), 2.79 (t, J=6.27 Hz, 4H), 2.33 (t, J=7.65 Hz, 2H), 2.07 (q,J=6.94 Hz, 8H), 1.83 (dddd, J=14.40, 8.69, 5.77, 3.26 Hz, 1H), 1.54-1.75(m, 3H), 1.42-1.54 (m, 3H), 1.23-1.42 (m, 32H), 0.85-0.96 (m, 6H)

Example 49:(9Z,12Z)-(12Z,15Z)-3-((3-(dimethylamino)propanoyl)oxy)henicosa-12,15-dien-1-yloctadeca-9,12-dienoate

Example 49 can be prepared using similar methods to those employed forthe synthesis of Example 1. ¹H NMR (400 MHz, CDCl₃) δ=5.16-5.40 (m, 8H),4.92 (s, 1H), 3.91-4.16 (m, 2H), 2.70 (t, J=6.3 Hz, 4H), 2.49-2.59 (m,2H), 2.36-2.45 (m, 2H), 2.21 (t, J=7.5 Hz, 2H), 2.18 (s, 6H), 1.98 (q,J=7.1 Hz, 8H), 1.74-1.86 (m, 2H), 1.43-1.59 (m, 4H), 1.14-1.35 (m, 32H),0.69-0.90 (m, 6H). MS (M+1)=686.5, Rt=1.13 min (LC Method 6).

The following examples (Examples 50-53) can be prepared using similarmethods to those employed for the synthesis of Example 49.

Example 50:(12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)henicosa-12,15-dien-1-yl3-octylundecanoate, formate salt

¹H NMR (400 MHz, CDCl₃) δ=8.53 (s, 1H), 5.27-5.47 (m, 4H), 4.93-5.05 (m,1H), 4.09 (t, J=6.65 Hz, 2H), 2.79 (t, J=6.80 Hz, 2H), 2.67 (dd, J=9.03,6.53 Hz, 2H), 2.50 (s, 6H), 2.39 (t, J=7.15 Hz, 2H), 2.23 (d, J=6.78 Hz,2H), 2.07 (q, J=6.80 Hz, 4H), 1.78-1.99 (m, 5H), 1.48-1.69 (m, 3H),1.20-1.44 (m, 46H), 0.83-0.96 (m, 9H). MS (M+1)=718.4, Rt=1.34 min (LCMethod 6).

Example 51:(12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)henicosa-12,15-dien-1-yl5-heptyldodecanoate

¹H NMR (400 MHz, CDCl₃) δ=5.23-5.49 (m, 4H), 4.84-5.08 (m, 1H),3.95-4.19 (m, 2H), 2.74-2.83 (m, 2H), 2.56 (br. s., 6H), 2.42 (t, J=6.82Hz, 2H), 2.27 (t, J=7.58 Hz, 2H), 2.06 (q, J=6.91 Hz, 6H), 1.77-1.97 (m,2H), 1.48-1.67 (m, 5H), 1.14-1.42 (m, 46H), 0.81-0.96 (m, 9H). MS(M+1)=718.5, Rt=1.25 min (LC Method 5).

Example 52:(12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)henicosa-12,15-dien-1-yl7-hexyltridecanoate

¹H NMR (400 MHz, CDCl₃) δ=5.25-5.48 (m, 4H), 4.91-5.06 (m, 1H),4.00-4.21 (m, 2H), 2.78 (t, J=6.53 Hz, 2H), 2.24-2.48 (m, 12H),1.99-2.13 (m, 4H), 1.71-1.99 (m, 7H), 1.48-1.71 (m, 4H), 1.24-1.48 (m,30H), 1.22 (br. s., 12H), 0.76-0.99 (m, 9H). ¹³C NMR (101 MHz, CDCl₃)δ=173.90, 173.10, 130.19, 130.10, 127.96 (2C), 71.12, 60.55, 58.82,45.26 (2C) 37.35, 34.32, 34.23, 33.60 (2C) 33.49, 33.02, 32.09, 31.94(2C) 31.51, 29.82 (3C), 29.66 (2C), 29.48 (2C), 29.34, 29.29, 27.22,27.18, 26.62 (2C), 26.37, 25.60, 25.19, 24.99, 22.71 (2C), 22.57, 22.54,14.14 (2C), 14.09.

Example 53:(12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)henicosa-12,15-dien-1-yl9-pentyltetradecanoate

¹H NMR (400 MHz, CDCl₃) δ=5.22-5.49 (m, 4H), 4.84-5.09 (m, 1H),3.96-4.21 (m, 2H), 2.67-2.83 (m, 3H), 2.57 (br. s., 6H), 2.42 (t, J=6.82Hz, 2H), 2.29 (t, J=7.58 Hz, 2H), 2.06 (q, J=6.91 Hz, 6H), 1.77-1.98 (m,2H), 1.48-1.67 (m, 4H), 1.14-1.43 (m, 46H), 0.82-0.97 (m, 9H). MS(M+1)=718.5, Rt=1.17 min (LC Method 6).

Synthesis of Example 54 Intermediate 54a:(9Z,12Z)-octadeca-9,12-dien-1-yl carbonochloridate

To a solution of linoleyl alcohol (2.0 g, 7.5 mmol) in 40 mLethylacetate, cooled in an ice-water bath, was added triphosgene (1.11g, 3.8 mmol), followed by DIPEA (2.1 ml, 12.0 mmol). The reaction waswarmed to room temperature and stirred for further 10 min. The reactionwas then filtered, concentrated under reduced pressure and purified onsilica gel with 20% DCM/heptane to afford desired product (2.0 g, 81%).¹H NMR (CDCl₃) δ=5.25-5.54 (m, 4H), 4.34 (t, J=6.8 Hz, 2H), 2.80 (t,J=6.4 Hz, 2H), 2.07 (q, J=6.9 Hz, 4H), 1.63-1.82 (m, 2H), 1.19-1.46 (m,16H), 0.81-0.99 (m, 3H)

Intermediate 54b: (12Z,15Z)-3-hydroxyhenicosa-12,15-dien-1-yl(9Z,12Z)-octadeca-9,12-dien-1-yl carbonate

To a solution of Intermediate 49b (50.0 mg, 0.15 mmol) in 2.0 ml DCM,cooled in an ice-water bath, was added Intermediate 54a (60.8 mg, 0.185mmol), followed by pyridine (48.7 mg, 0.616 mmol). The reaction wasstirred at for 4.5 hrs. The reaction was quenched with sat. aq. NaHCO₃and extracted with ethyl acetate. The combined organics were dried oversodium sulfate and filtered. The filtrate was concentrated under reducedpressure and the residue was purified on silica gel with 15%ethylacetate/heptane to afford the title compound. ¹H NMR (CDCl₃)δ=5.27-5.57 (m, 8H), 4.34-4.49 (m, 1H), 4.20-4.32 (m, 1H), 4.15 (t,J=6.7 Hz, 2H), 3.75 (d, J=6.1 Hz, 1H), 2.80 (t, J=6.4 Hz, 4H), 2.07 (q,J=6.8 Hz, 8H), 1.82-1.96 (m, 1H), 1.57-1.78 (m, 6H), 1.22-1.52 (m, 34H),0.84-0.98 (m, 6H)

Example 54:(12Z,15Z)-1-((((9Z,12Z)-octadeca-9,12-dien-1-yloxy)carbonyl)oxy)henicosa-12,15-dien-3-yl3-(dimethylamino)propanoate

Example 54 can be prepared using similar methods to those employed forthe synthesis of Example 49. 1H NMR (CDCl₃) δ=5.26-5.51 (m, 8H), 5.02(br. s., 1H), 4.15 (dt, J=19.7, 6.7 Hz, 4H), 2.79 (t, J=6.4 Hz, 4H),2.57-2.67 (m, 2H), 2.41-2.57 (m, 2H), 2.26 (s, 6H), 2.06 (q, J=6.9 Hz,8H), 1.86-2.00 (m, 2H), 1.64-1.73 (m, 2H), 1.23-1.42 (m, 36H), 0.85-0.97(m, 6H). MS (M+1)=716.8. Rt=1.12 min. (LC Method 11).

Synthesis of Example 55 Intermediate 55a: heptyl2,2-bis(heptyloxy)acetate

To a solution of methyl 2,2-dimethoxyacetate (5.0 g, 37.3 mmol) inheptanol (26.3 mL, 186 mmol) was added camphorsulfonic acid (0.43 g,1.86 mmol), and the reaction was heated to 100° C., overnight. Thereaction was cooled to ambient temperature and concentrated underreduced pressure. The concentrate was purified on silica gel withdichloromethane/heptane as eluent to provide 4.0 g of the desiredcompound. ¹H NMR (400 MHz, CDCl₃): δ=4.86 (s, 1H), 4.21 (t, J=6.78 Hz,2H), 3.52-3.69 (m, 4H), 1.55-1.77 (m, 6H), 1.20-1.45 (m, 24H), 0.82-0.98(m, 9H) ppm.

Intermediate 55b: 2,2-bis(heptyloxy)acetic acid

To a solution of Intermediate 26a (4.06 g, 10.5 mmol) in methanol (50mL) was added sodium hydroxide (2N aq, 7.88 mL, 15.8 mmol), and themixture was stirred at ambient temperature overnight. The reaction wasdiluted with ethyl acetate and brine. The aqueous layer was titrated toneutral pH with 1N aq HCl, and was extracted with ethyl acetate. Thecombined organics were dried over sodium sulfate, filtered, andconcentrated under reduced pressure. The concentrate was purified onsilica gel (equilibrated with 0.4N ammonia and 2% methanol indichloromethane) with methanol/dichloromethane as eluent to provide 2.3g of the desired compound. ¹H NMR (400 MHz, CDCl₃): δ=4.88 (s, 1H),3.53-3.71 (m, 4H), 1.63 (quin, J=6.84 Hz, 4H), 1.18-1.43 (m, 16H),0.84-0.97 (m, 6H) ppm.

Intermediate 55c

Example 55c can be prepared using similar methods to those employed forthe synthesis of Intermediate 49c. ¹H NMR (400 MHz, CDCl₃) δ=5.30-5.46(m, 4H), 4.90 (s, 1H), 4.17-4.30 (m, 2H), 3.53-3.70 (m, 5H), 2.80 (t,J=6.44 Hz, 2H), 2.07 (q, J=6.82 Hz, 4H), 1.71-1.96 (m, 2H), 1.59-1.70(m, 4H), 1.42-1.54 (m, 4H), 1.19-1.42 (m, 34H), 0.80-0.99 (m, 9H)

Example 55:(13Z,16Z)-4-(((2-(dimethylamino)ethoxy)carbonyl)oxy)docosa-13,16-dien-1-yl2,2-bis(heptyloxy)acetate

To a solution of Intermediate 55c (174 mg, 0.286 mmol) in DCM (4.0 mL)cooled in an ice-water bath, was added pyridine (0.035 mL, 0.429 mmol)followed by triphosgene (42.4 mg, 0.143 mmol). After 30 min,2-(dimethylamino)ethanol (0.086 mL, 0.857 mmol) was added and theresultant mixture was stirred at room temperature for 1 hr. The reactionwas extracted between sat. NaHCO₃ and DCM. The combined organics weredried over sodium sulfate and filtered. The filtrate was concentratedunder reduced pressure and the residue purified on silica gel with 60%ethyl acetate/heptane eluent to provide the title compound. ¹H NMR(CDCl₃) δ=5.19-5.37 (m, 4H), 4.81 (s, 1H), 4.64 (s, 1H), 4.07-4.18 (m,4H), 3.43-3.59 (m, 4H), 2.66-2.74 (m, 2H), 2.52 (t, J=5.9 Hz, 2H), 2.21(s, 6H), 1.92-2.02 (m, 4H), 1.38-1.76 (m, 12H), 1.12-1.36 (m, 32H),0.72-0.88 (m, 9H). MS (M+1)=724.4, Rt=1.21 min (LC Method 6)

Example 56:(13Z,16Z)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)docosa-13,16-dien-1-yl2,2-bis(heptyloxy)acetate

¹H NMR (CDCl₃) δ=5.25-5.51 (m, 4H), 4.89 (s, 1H), 4.61-4.79 (m, 1H),4.09-4.29 (m, 4H), 3.44-3.71 (m, 4H), 2.79 (t, J=6.4 Hz, 2H), 2.45-2.61(m, 6H), 2.00-2.15 (m, 4H), 1.71-1.89 (m, 4H), 1.47-1.71 (m, 8H),1.24-1.43 (m, 34H), 1.02 (t, J=7.2 Hz, 6H), 0.86-0.95 (m, 9H). MS(M+1)=766.5, Rt=1.22 min (LC Method 6).

Synthesis of Example 57 Intermediate 57a:((2,2-bis(heptyloxy)ethoxy)methyl)benzene

Benzyloxylacetaldehyde (2 g, 13.3 mmol), heptanol (4.64 g, 40.0 mmol)and PPTS (33.0 mg, 0.13 mmol) were combined in a round bottom flask. Themixture was heated at 110° C. for 72 h. The reaction was directly loadedonto silica gel and purified with 3% ethyl acetate/heptane to afford thedesired product (3.0 g, 62%). ¹H NMR (CDCl₃) δ=7.30-7.40 (m, 5H), 4.68(t, J=5.3 Hz, 1H), 4.61 (s, 2H), 3.61-3.66 (m, 2H), 3.48-3.55 (m, 4H),1.60-1.72 (m, 4H), 1.27-1.38 (m, 16H), 0.87-0.93 (m, 6H).

Intermediate 57b: 2,2-bis(heptyloxy)ethanol

To a solution of Intermediate 57a (2.7 g, 7.41 mmol) in 40 ml EtOAc/MeOH(1:1) at room temperature, 10% Pd/C (0.788 g 0.74 mmol) was added. Thereaction atmosphere was exchanged with H₂ and treated with an H₂ ballonat room temperature for 3 h. The reaction was filtered, concentratedunder reduced pressure, and purified on silica gel with 12%ethylacetate/heptane to afford the desired product (1.0 g, 49%). ¹H NMR(CDCl₃) δ=4.55 (t, J=5.4 Hz, 1H), 3.70 (dt, J=9.3, 6.8 Hz, 2H), 3.59 (d,J=4.8 Hz, 2H), 3.51 (dt, J=9.3, 6.8 Hz, 2H), 1.61 (dt, J=14.3, 6.9 Hz,4H), 1.21-1.43 (m, 16H), 0.83-0.96 (m, 6H).

Intermediate 57c:3-((3-(2,2-bis(heptyloxy)ethoxy)-3-oxopropyl)disulfanyl)propanoic acid

To a suspension of disulfidedipropanoic acid (115 mg, 0.547 mmol) in 3.0ml DCM, Intermediate 57b (100 mg, 0.364 mmol), DMAP (17.81 mg, 0.656mmol) and DIPEA (32 ul, 0.182 mmol) were added. EDC (126 mg, 0.656 mmol)was added last. The mixture became clear and was stirred at roomtemperature for 1 hr. The reaction was extracted between brine and ethylacetate. The combined organics were dried over sodium sulfate,concentrated under reduced pressure and purified on silica gel with 30%ethyl acetate/heptane to afford the desired product (75 mg, 44%). ¹H NMR(CDCl₃) δ=4.72 (t, J=5.4 Hz, 1H), 4.15 (d, J=5.3 Hz, 2H), 3.65 (dt,J=9.3, 6.8 Hz, 2H), 3.51 (dt, J=9.3, 6.8 Hz, 2H), 2.90-3.03 (m, 4H),2.75-2.86 (m, 4H), 1.60 (quin, J=7.0 Hz, 4H), 1.19-1.43 (m, 16H),0.81-0.97 (m, 6H).

Example 57: 2,2-bis(heptyloxy)ethyl3-((3-ethyl-10-((9Z,12Z)-octadeca-9,12-dien-1-yl)-8,15-dioxo-7,9,14-trioxa-3-azaheptadecan-17-yl)disulfanyl)propanoate

Example 57 can be prepared using similar methods to those employed forthe synthesis of Example 55. ¹H NMR (CDCl₃) δ=5.23-5.44 (m, 4H),4.65-4.74 (m, 1H), 4.63 (t, J=5.4 Hz, 1H), 4.14 (td, J=6.5, 1.8 Hz, 2H),4.00-4.11 (m, 4H), 3.59 (dt, J=9.2, 6.7 Hz, 2H), 3.44 (dt, J=9.1, 6.7Hz, 2H), 2.83-2.95 (m, 4H), 2.65-2.79 (m, 6H), 2.41-2.52 (m, 6H), 2.01(q, J=6.9 Hz, 4H), 1.73-1.84 (m, 2H), 1.45-1.73 (m, 10H), 1.16-1.38 (m,34H), 0.97 (t, J=7.2 Hz, 6H), 0.78-0.89 (m, 9H). MS (M+1)=944.5, Rt=1.25min (LC Method 6).

Synthesis of Example 58 Example 58a

In a 500 ml round-bottom flask equipped with a stir bar, 9-heptadecanol(1.12 g, 4.37 mmol) was dissolved in DCM (30 ml).4-(tert-butoxy)-4-oxobutanoic acid (0.913 g, 5.24 mmol) and DMAP (0.107g, 0.873 mmol) were added, followed by DIPEA (3.05 ml, 17.47 mmol). Themixture is stirred at rt for a minute before addition of EDC.HCl (1.172g, 6.11 mmol). The mixture is then stirred at rt overnight. The mixturewas concentrated under reduced pressure, and the concentrate waspurified on silica gel with ethyl acetate/heptane as eluent to providethe title compound. ¹H NMR (400 MHz, CDCl₃) δ=4.89 (quin, J=6.27 Hz,1H), 2.48-2.65 (m, 4H), 1.48-1.57 (m, 4H), 1.42-1.48 (m, 9H), 1.18-1.38(m, 24H), 0.84-0.96 (m, 6H)

Example 58b

Intermediate 58a was treated with TFA (3.36 ml, 43.7 mmol), and stirredat rt for ˜30 min. The reaction was concentrated to dryness underreduced pressure to provide the title compound, which was used withoutfurther purification. ¹H NMR (400 MHz, CDCl₃) δ=7.94 (br. s., 1H), 4.91(quin, J=6.27 Hz, 1H), 2.54-2.83 (m, 4H), 1.50-1.62 (m, 4H), 1.18-1.39(m, 24H), 0.82-1.00 (m, 6H)

Example 58:(13Z,16Z)-4-(((3-(dimethylamino)propoxy)carbonyl)oxy)docosa-13,16-dien-1-ylheptadecan-9-yl succinate

¹H NMR (400 MHz, CDCl₃) δ=5.46-5.26 (m, 4H), 4.87 (t, J=6.1 Hz, 1H),4.72 (br. s., 1H), 4.19 (t, J=6.5 Hz, 2H), 4.14-4.04 (m, 2H), 2.78 (t,J=6.5 Hz, 2H), 2.62 (s, 4H), 2.43 (d, J=6.5 Hz, 2H), 2.29 (s, 6H), 2.05(q, J=7.0 Hz, 4H), 1.97-1.83 (m, 2H), 1.79-1.44 (m, 10H), 1.42-1.17 (m,42H), 0.95-0.82 (m, 9H) ppm. LC-MS m/z=807.2 (MH+). Rt=2.01 min (LCMethod 11).

Synthesis of Example 59 Intermediate 59a:2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)acetaldehyde

To a solution of DMSO (1.04 ml, 14.7 mmol) in 35 mL DCM, cooled in adry-ice/acetone bath, was added oxalyl chloride (0.96 ml, 11.03 mmol),dropwise. After 30 min., a solution of2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)ethanol (1.62 g, 7.35 mmol) in5 ml DCM was added dropwise. The reaction stirred for 45 min. and TEA(5.12 ml, 36.8 mmol) was added. After 15 min. the reaction was warmed toroom temperature. Sat. aq. NH₄Cl was added and the reaction wasextracted with ethyl acetate. The combined organics were dried oversodium sulfate, concentrated under reduced pressure and purified onsilica gel with 30% EtOAc/hexane to afford the desired product (1.0 g,62%). ¹H NMR (400 MHz, CDCl₃) δ=9.67 (s, 1H), 4.10 (d, J=1.0 Hz, 2H),3.73-3.77 (m, 2H), 3.56-3.60 (m, 2H), 0.81-0.83 (m, 9H), −0.02 (s, 6H).

Intermediate 59b:(11Z,14Z)-1-(2-((tert-butyldimethylsilyl)oxy)ethoxy)icosa-11,14-dien-2-ol

To a solution of Intermediate 59a (0.78 g, 3.57 mmol) in 6.0 ml THF,cooled in an ice-water bath, was added linoleylmagnesium bromide (12.50ml, 0.4M THF solution, 5.0 mmol), dropwise. The reaction was stirred for30 min. The reaction was quenched with Sat. aq. NaHCO₃ and extractedwith ethyl acetate. The combined organics were dried over sodiumsulfate, concentrated under reduced pressure, and purified on silica gelwith 10% ethyl acetate/heptane to afford the desired product (0.85 g,51%). ¹H NMR (400 MHz, CDCl₃) δ=5.17-5.40 (m, 4H), 3.63-3.77 (m, 3H),3.42-3.57 (m, 3H), 3.22 (dd, J=12.0, 8.0 Hz, 1H), 2.69 (m, 2H), 1.97 (m,4H), 1.11-1.43 (m, 21H), 0.74-0.86 (m, 12H), −0.05-0.04 (s, 6H).

Intermediate 59c:(11Z,14Z)-1-(2-((tert-butyldimethylsilyl)oxy)ethoxy)icosa-11,14-dien-2-yl3-(dimethylamino)propanoate

To a solution of Intermediate 59b (450 mg, 0.96 mmol) anddimethylaminopropanoic acid (188 mg, 1.15 mmol) in 10 ml DCM, were addedDMAP (46.9 mg, 0.38 mmol) and DIPEA (0.50 ml, 2.88 mmol). EDC (258 mg,1.15 mmol) was added last. The resulting mixture was stirred at roomtemperature overnight. The reaction was extracted between brine andethyl acetate. The combined organics were dried over sodium sulfate,concentrated under reduced pressure, and purified on silica gel with 6%MeOH/DCM to afford the desired product (370 mg, 68%). ¹H NMR (400 MHz,CDCl₃) δ=5.20-5.43 (m, 4H), 4.91-5.04 (m, 1H), 3.61-3.73 (m, 2H),3.38-3.56 (m, 4H), 2.71 (t, J=4.0 Hz, 2H), 2.53-2.64 (m, 2H), 2.38-2.50(m, 2H), 2.20 (s, 6H), 1.92-2.05 (m, 4H), 1.12-1.35 (m, 20H), 0.76-0.88(m, 12H), 0.00 (s, 6H).

Intermediate 59d: (11Z,14Z)-1-(2-hydroxyethoxy)icosa-11,14-dien-2-yl3-(dimethylamino)propanoate

To a solution of Intermediate XXc (370 mg, 0.651 mmol) in 10 ml THF,TBAF (0.717 ml 1.0M in THF solution, 0.717 mmol) was added dropwise. Theresulting mixture was stirred at room temperature for 2 h. The reactionwas extracted between brine and ethyl acetate. The combined organicswere dried over sodium sulfate, filtered, and concentrated under reducedpressure to provide the desired product, which was used without furtherpurification. ¹H NMR (400 MHz, CDCl₃) δ=5.26 (s, 4H), 4.93-5.11 (m, 1H),3.55-3.63 (m, 2H), 3.46-3.54 (m, 1H), 3.39-3.46 (m, 3H), 2.39-2.82 (m,6H), 2.27 (s, 6H), 1.89-2.00 (m, 4H), 1.10-1.29 (m, 20H), 0.77-0.81 (m,3H).

Example 59:(9Z,12Z)-2-(((11Z,14Z)-2-((3-(dimethylamino)propanoyl)oxy)icosa-11,14-dien-1-yl)oxy)ethyloctadeca-9,12-dienoate

Example 59 can be prepared using similar methods to those employed forthe synthesis of Example 1. ¹H NMR (CDCl₃) δ=5.18-5.62 (m, 8H), 5.04 (s,1H), 4.21 (t, J=4.9 Hz, 2H), 3.68-3.79 (m, 1H), 3.60-3.68 (m, 1H), 3.54(t, J=4.5 Hz, 2H), 2.79 (t, J=6.3 Hz, 4H), 2.60-2.69 (m, 2H), 2.46-2.55(m, 2H), 2.33 (t, J=7.6 Hz, 2H), 2.26 (s, 6H), 2.06 (q, J=7.2 Hz, 8H),1.54-1.71 (m, 4H), 1.22-1.44 (m, 32H), 0.85-0.99 (m, 6H). MS(M+1)=717.1, Rt=1.32 min (LC Method 6).

Synthesis of Example 60 Intermediate 60a:2-((10-bromodecyl)oxy)tetrahydro-2H-pyran

To a solution of 10-bromo-1-decanol (85% purity, 6.0 g, 25.3 mmol) indichloromethane was added dihydropyran (2.75 mL, 30.4 mmol) andp-toluenesulfonic acid monohydrate (2.5 g, 12.6 mmol). The resultingmixture was stirred at 30° C. for 2 h. The reaction was diluted withwater and extracted with dichloromethane (2×50 mL). The combineddichloromethane extracts were washed with brine (50 mL), dried oversodium sulfate, and filtered. The filtrate was concentrated underreduced pressure and the crude material was purified on silica gel withethyl acetate/hexanes as eluent to provide the desired product. TLC(silica gel, 20% ethyl acetate:hexane): R_(f)=0.73.

Intermediate 60b: 2-((10-iododecyl)oxy)tetrahydro-2H-pyran

To a solution of Intermediate 60a (6.0 g, 18.74 mmol) in acetone (80 mL)was added sodium iodide (8.4 g, 56.2 mmol). The reaction was heated toreflux for 24 h. the reaction mixture was cooled to ambient temperatureand concentrated under reduced pressure. The concentrate was dilutedwith water (100 mL) and extracted with dichloromethane (2×50 mL). Thecombined dichloromethane extracts were washed with brine (100 mL), driedover sodium sulfate, and filtered. The filtrate was concentrated underreduced pressure to provide the desired product, which was used withoutfurther purification. TLC (silica gel, 20% ethyl acetate:hexane):R_(f)=0.73.

Intermediate 60c:1-((tert-butyldimethylsilyl)oxy)-13-((tetrahydro-2H-pyran-2-yl)oxy)tridecan-3-ol

To a solution of Intermediate 60b (6.00 g, 16.30 mmol) in pentane (75mL) and diethyl ether (25 mL), under an argon atmosphere and cooled in adry-ice/acetone bath, was added tert-butyl lithium (1.5M in pentane, 22mL, 32.6 mmol). A solution of 3-((tert-butyldimethylsilyl)oxy) propanalin diethyl ether (20 mL) was added. The reaction was quenched withaqueous saturated ammonium chloride (50 mL), and the reaction wasallowed to warm to ambient temperature. The aqueous layer was extractedwith diethyl ether (2×100 mL). The combined organic extracts were driedover sodium sulfate and filtered. The filtrate was concentrated underreduced pressure, and the crude material was purified on silica gel withethyl acetate/hexanes as eluent to provide the desired product. TLC(silica gel, 10% ethyl acetate:hexane): R_(f)=0.19.

Intermediate 60d:1-((tert-butyldimethylsilyl)oxy)-13-((tetrahydro-2H-pyran-2-yl)oxy)tridecan-3-yl(4-nitrophenyl) carbonate

To a solution of Intermediate 60c (2.5 g, 5.81 mmol) in dichloromethane(20 mL) was added 4-nitrophenyl chloroformate (1.75 g, 8.71 mmol) andpyridine (1.5 mL, 17.43 mmol). The reaction was stirred at 30° C. for 2h. The reaction mixture was diluted with water (50 mL) and the aqueouslayer was extracted with dichloromethane (2×50 mL). the combineddichloromethane extracts were washed with brine (50 mL), dried oversodium sulfate, and filtered. The filtrate was concentrated underreduced pressure, and the crude material was purified on silica gel withethyl acetate/hexanes as eluent to provide the desired product. TLC(silica gel, 20% ethyl acetate:hexane): R_(f)=0.73.

Intermediate 60e:1-((tert-butyldimethylsilyl)oxy)-13-((tetrahydro-2H-pyran-2-yl)oxy)tridecan-3-yl(3-(dimethylamino)propyl) carbonate

To a solution of Intermediate 60d (2.6 g, 4.37 mmol) in dichloromethanewas added 2-(dimethylamino)-1-propanol (1.0 mL, 8.73 mmol), pyridine(1.1 mL, 13.3 mmol), and DMAP (0.53 g, 4.37 mmol). The resulting mixturewas stirred at 30° C. for 8 h, then additional2-(dimethylamino)-1-propanol (1.0 mL, 8.73 mmol), pyridine (1.1 mL, 13.3mmol), and DMAP (0.53 g, 4.37 mmol) were added. The reaction was stirredfor an additional 16 h, then additional 2-(dimethylamino)-1-propanol(1.0 mL, 8.73 mmol), pyridine (1.1 mL, 13.3 mmol), and DMAP (0.53 g,4.37 mmol) were added. The reaction was stirred for an additional 24 h.The reaction mixture was diluted with water (100 mL), and the aqueouslayer was extracted with dichloromethane (2×100 mL). The combineddichloromethane extracts were washed with brine (100 mL), dried oversodium sulfate, and filtered. The filtrate was concentrated underreduced pressure, and the crude material was purified on silica gel withmethanol/dichloromethane as eluent to provide the desired product. TLC(silica gel, 10% methanol:dichloromethane): R_(f)=0.45.

Intermediate 60f: 3-(dimethylamino)propyl(1-hydroxy-13-((tetrahydro-2H-pyran-2-yl)oxy)tridecan-3-yl) carbonate

To a mixture of ceric ammonium nitrate (800 mg, 1.46 mmol) in water (20mL) was added 50% pyridine in water to titrate the solution to pH=6. Asolution of Intermediate 60e (1.7 g, 3.04 mmol) in THF (30 mL) wasadded, and the mixture was stirred at 30° C. for 24 h. The reaction wasdiluted with water (30 mL) and extracted with ethyl acetate (2×50 mL).The combined organic extracts were washed with brine (50 mL), dried oversodium sulfate, and filtered. The filtrate was concentrated underreduced pressure to provide the desired product, which was used withoutfurther purification. TLC (silica gel, 10% methanol:dichloromethane):R_(f)=0.24.

Intermediate 60g:(9Z,12Z)-3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-((tetrahydro-2H-pyran-2-yl)oxy)tridecyloctadeca-9,12-dienoate

To a solution of Intermediate 60f (1.2 g, 2.69 mmol) in dichloromethane(50 mL), was added linoleic acid (1.13 g, 4.04 mmol), EDC.HCl (1.03 g,5.39 mmol), DIPEA (1.4 mL, 8.09 mmol), and DMAP (165 mg, 1.35 mmol). Theresulting mixture was stirred at 30° C. for 16 h. The reaction mixturewas diluted with water (50 mL), and the aqueous layer was extracted withdichloromethane (2×100 mL). The combined dichloromethane extracts werewashed with brine (100 mL), dried over sodium sulfate, and filtered. Thefiltrate was concentrated under reduced pressure, and the crude materialwas purified on silica gel with methanol/dichloromethane as eluent toprovide the desired product. TLC (silica gel, 10%methanol:dichloromethane): R_(f)=0.67

Intermediate 60h:(9Z,12Z)-3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-hydroxytridecyloctadeca-9,12-dienoate

To a solution of Intermediate 60g (500 mg, 0.706 mmol) in THF (10 mL),cooled in an ice-water bath, was added 6N HCl (aq, 10 mL). the resultingmixture was stirred for 2 h, then warmed to ambient temperature. Thereaction was neutralized with saturated sodium bicarbonate solution, andthe reaction was extracted with ethyl acetate (2×40 mL). The combinedorganic extracts were washed with brine, dried over sodium sulfate, andfiltered. The filtrate was concentrated under reduced pressure, and thecrude material was purified on silica gel with methanol/dichloromethaneas eluent to provide the desired product. TLC (silica gel, 10%methanol:dichloromethane): R_(f)=0.39

Example 60:(9Z,12Z)-3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyloctadeca-9,12-dienoate

To a solution of octanoic acid (320 mg, 0.513 mmol) in dichloromethane(20 mL) was added EDC.HCl (295 mg, 1.54 mmol) and DIPEA (0.45 mL, 2.57mmol). The reaction was stirred at 30° C. for 1 h, then Intermediate 60h(320 mg, 0.513 mmol) and DMAP (63 mg, 0.513 mmol) were added and thereaction stirred for an additional 23 h. the reaction was diluted withwater (20 mL) and the aqueous layer was extracted with dichloromethane(2×30 mL). the combined dichloromethane extracts were washed with brine(20 mL), dried over sodium sulfate and filtered. The filtrate wasconcentrated under reduced pressure, and the crude material was purifiedon silica gel with methanol/dichloromethane as eluent to provide thedesired product. ¹H NMR (400 MHz, CDCl₃) δ=5.27-5.47 (m, 4H) 4.81 (t,J=6.19 Hz, 1H) 4.10-4.25 (m, 4H) 4.06 (t, J=6.82 Hz, 2H) 2.78 (t, J=6.57Hz, 2H) 2.16-2.45 (m, 10H) 2.06 (q, J=6.82 Hz, 4H) 1.77-1.99 (m, 4H)1.48-1.71 (m, 8H) 1.24-1.44 (m, 38H) 0.81-0.96 (m, 6H). MS (M+1)=750.7,Rt=1.25 min (LC Method 5).

Example 61:3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl3-octylundecanoate

¹H NMR (400 MHz, CDCl₃) δ=4.81 (quin, J=6.25 Hz, 1H) 4.16-4.26 (m, 2H)4.09-4.16 (m, 2H) 4.06 (t, J=6.69 Hz, 2H) 2.41 (br. s., 2H) 2.19-2.36(m, 8H) 1.77-2.01 (m, 5H) 1.50-1.74 (m, 10H) 1.14-1.41 (m, 48H)0.77-1.00 (m, 9H). ¹³C NMR (101 MHz, CDCl₃) δ=173.68, 173.23, 154.61,75.35, 65.89, 64.05, 60.12, 55.64, 44.97 (2C), 38.89, 34.69, 34.09,33.90, 33.55 (2C), 32.74, 31.58 (4C), 31.34, 29.60 (2C), 29.28 (2C),29.17 (2C), 29.00 (2C), 28.93, 28.79, 28.60, 28.35, 26.22 (3C), 25.63,24.75, 24.70, 22.35 (2C), 22.26, 13.78 (2C), 13.73.

Example 62:3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-hydroxytridecyl5-heptyldodecanoate

¹H NMR (400 MHz, CDCl₃) δ=4.74 (quin, J=6.50 Hz, 1H), 4.12 (t, J=6.40Hz, 2H), 4.01-4.09 (m, 2H), 3.57 (t, J=6.53 Hz, 2H), 2.46 (br. s, 2H),2.29 (br. s., 6H), 2.20 (t, J=7.65 Hz, 2H), 1.80-1.95 (m, 4H), 1.44-1.63(m, 6H), 1.38 (br. s, 1H), 1.07-1.33 (m, 41H), 0.75-0.87 (m, 6H). 13CNMR (101 MHz, CDCl₃): Q=173.87, 154.89, 75.72, 65.93, 63.03, 60.48,55.87, 44.93 (2C), 37.23, 34.69, 34.19, 33.48 (2C), 33.18, 33.02, 32.80,31.96 (2C), 30.11, 29.72, 29.52, 29.45-29.35 (6C), 26.66 (2C), 26.34,25.72, 25.01, 22.72 (2C), 22.17, 14.17 (2C).

Example 63:3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl5-heptyldodecanoate

¹H NMR (400 MHz, CDCl₃) δ=4.73 (quin, J=6.20 Hz, 1H), 4.12 (t, J=6.40Hz, 2H), 4.02-4.09 (m, 2H), 3.98 (t, J=6.78 Hz, 2H), 2.34-2.59 (m, 2H),2.26 (br. s., 6H), 2.21 (q, J=7.19 Hz, 4H), 1.78-1.92 (m, 4H), 1.44-1.65(m, 8H), 1.10-1.30 (m, 49H), 0.81 (quin, J=6.50 Hz, 9H). MS (M+1)=768.7,Rt=1.23 min (LC Method 6).

Example 64:3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl7-hexyltridecanoate

¹H NMR (400 MHz, CDCl₃) δ=4.89-4.76 (m, 1H), 4.24-4.10 (m, 4H), 4.06 (t,J=6.8 Hz, 2H), 2.44-2.33 (m, 2H), 2.30 (t, J=7.6 Hz, 4H), 2.23 (s, 6H),1.98-1.77 (m, 4H), 1.73-1.48 (m, 9H), 1.42-1.15 (m, 48H), 0.97-0.79 (m,9H). ¹³C NMR (101 MHz, CDCl₃) δ=173.99, 173.76, 154.97, 75.62, 66.34,64.37, 60.55, 56.00, 45.46 (2C), 37.40, 34.40, 34.30, 34.22, 33.66 (2C),33.55, 33.02, 31.95 (2C), 31.66, 29.81 (2C), 29.68, 29.49 (4C), 29.25,29.11, 28.92, 28.67, 27.00, 26.65 (2C), 26.40, 25.94, 25.07, 25.02,24.98, 22.70 (2C), 22.58, 14.11 (2C), 14.05.

Example 65:3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-hydroxytridecyl9-pentyltetradecanoate

¹H NMR (400 MHz, CDCl₃) δ=4.74 (quin, J=6.21 Hz, 1H), 4.11 (td, J=6.50,1.76 Hz, 2H), 4.06 (t, J=6.50 Hz, 2H), 3.57 (t, J=6.65 Hz, 2H), 2.37 (t,J=6.78 Hz, 2H), 2.22 (s, 6H), 1.77-1.89 (m, 4H), 1.43-1.62 (m, 6H),1.07-1.33 (m, 43H), 0.81 (t, J=7.03 Hz, 6H). MS (M+1)=642.5, Rt=0.95 min(LC Method 6).

Example 66:3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl9-pentyltetradecanoate

¹H NMR (400 MHz, CDCl₃) δ=4.81 (quin, J=6.25 Hz, 1H) 4.19 (t, J=6.57 Hz,2H) 4.14 (t, J=6.57 Hz, 2H) 4.06 (t, J=6.69 Hz, 2H) 2.35-2.55 (m, 2H)2.19-2.35 (m, 9H) 1.80-1.99 (m, 4H) 1.49-1.73 (m, 12H) 1.13-1.42 (m,46H) 0.79-0.98 (m, 9H). MS (M+1)=769.0, Rt=3.11 min (LC Method 4).

Example 67:1-(3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl)10-octyl decanedioate

¹H NMR (400 MHz, CDCl₃) δ=4.71-4.89 (m, 1H), 4.18 (t, J=6.53 Hz, 2H),4.08-4.16 (m, 2H), 4.05 (t, J=6.78 Hz, 4H), 2.44 (t, J=7.15 Hz, 2H),2.21-2.35 (m, 12H), 1.81-2.01 (m, 4H), 1.56-1.73 (m, 11H), 1.54 (br. s.,1H), 1.18-1.41 (m, 40H), 0.78-0.95 (m, 6H). MS (M+1)=784.5, Rt=0.99 min(LC Method 6).

Example 68:3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl10-(octanoyloxy)decanoate

¹H NMR (400 MHz, CDCl₃) δ=4.67-4.92 (m, 1H), 4.29 (ddd, J=11.31, 6.63,4.80 Hz, 1H), 4.13-4.24 (m, 2H), 3.98-4.13 (m, 5H), 3.20 (t, J=6.57 Hz,2H), 2.89 (s, 6H), 2.29 (t, J=7.45 Hz, 6H), 2.09-2.23 (m, 2H), 1.80-2.05(m, 2H), 1.50-1.74 (m, 12H), 1.28 (d, J=8.59 Hz, 40H), 0.77-0.98 (m,6H). MS (M+1)=784.6, Rt=1.11 min (LC Method 6).

Synthesis of Example 69 Intermediate 69a

To a solution of Intermediate 24c (2.75 g, 9.21 mmol) in THF (30.7 mL)was added lithium aluminum hydride (1M in diethyl ether, 10.1 mL, 10.1mmol) dropwise. The reaction was stirred at ambient temperature for 1.5hr. Several drops of EtOAc were added to quench the reaction, followedby sat. aq. NH₄Cl. The reaction was filtered with ethyl acetate washes,and the filtrate was concentrated under reduced pressure to provide 2.4g of the title compound, which was used without further purification. ¹HNMR (400 MHz, CDCl₃) δ=3.67 (t, J=6.95 Hz, 2H), 1.48-1.59 (m, 2H), 1.42(br. s., 1H), 1.18-1.37 (m, 29H), 0.83-0.96 (m, 6H)

Intermediate 69b: 3-octylundecanal

TEA (5.31 ml, 38.3 mmol) was added dropwise via syringe to a solution ofIntermediate 69a (3.63 g, 12.76 mmol) in DCM (100 ml) and cooled in anice-water bath. In a separate flask, SO₃.Py (3.05 g, 19.14 mmol) andDMSO (8.16 ml, 115 mmol) were added together and this mixture was addedto the first rbf slowly via syringe. The reaction was stirred for 30min, then the ice bath was removed. The reaction was stirred at ambienttemperature for an additional 24 hr. The reaction mixture was quenchedwith water. The DCM layer was collected, and the water was re-extractedwith EtOAc×1. The organic layers were combined, dried over MgSO₄,filtered, and concentrated under reduced pressure. The crude oil wasfiltered over a silica gel plug with heptanes (250 ml). The solventswere removed under reduced pressure to provide compound the titlecompound as a colorless oil (3.3 g, 91%), which was used without furtherpurification. ¹H NMR (400 MHz, CDCl₃) δ=9.77 (t, J=2.40 Hz, 1H), 2.33(dd, J=6.57, 2.27 Hz, 2H), 1.95 (br. s., 1H), 1.17-1.43 (m, 28H),0.81-0.97 (m, 6H)

Intermediate 69c: 5-octyltridec-1-en-3-ol

Vinyl magnesium bromide (12.85 mL, 12.85 mmol) was added dropwise viasyringe to a round bottom flask charged with anhydrous THF (75 ml),cooled in an ice-water bath and under N₂. Intermediate 69b (3.3 g, 11.68mmol) was slowly added as a solution in 25 mL of THF via syringe. Thereaction was stirred for 1.5 h. maintained in the ice-water bath, thereaction was quenched with sat. aq. NH₄Cl, and filtered over celite withEtOAc washes. The filtrate was poured into a sep funnel, and dilutedfurther with EtOAc. The layers were separated and the aqueous layer wasextracted with EtOAc once more. The combined organics were dried overNa₂SO₄, filtered, and the filtrate was concentrated under reducedpressure. The crude oil was purified on silica gel with 0-50%EtOAc/heptanes to provide the title compound as a colorless oil (2.8 g,77%). ¹H NMR (400 MHz, CDCl₃) δ=5.87 (ddd, J=17.05, 10.48, 6.32 Hz, 1H),5.23 (dt, J=17.18, 1.39 Hz, 1H), 5.10 (dt, J=10.36, 1.26 Hz, 1H),4.13-4.24 (m, 1H), 1.36-1.59 (m, 3H), 1.18-1.36 (m, 28H), 0.79-0.98 (m,6H).

Intermediate 69d:5-octyl-1-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)tridecan-3-ol

[Ir(cod)Cl]₂ (0.073 g, 0.161 mmol) and DPPE (0.096 g, 0.242 mmol), weredissolved in DCM in a round bottom flask charged with nitrogen.Pinacolborane (1.402 mL, 9.66 mmol) was added, followed by a solution ofIntermediate 69c (2.5 g, 8.05 mmol) in 5 ml DCM. After 4 hr of stirringat ambient temperature, another 1.5 ml of pinacolboronate was added. Thereaction was stirred overnight. Several water droplets were addedcarefully to quench the reaction. The reaction was diluted with DCM andwashed with aqueous sodium bicarbonate solution. The aqueous layer wasback extracted with DCM×1. The combined organics were dried over sodiumsulfate, filtered, and concentrated under reduced pressure. The crudematerial was purified on silica gel with 0-30% EtOAc/heptanes to providethe title compound as a colorless oil (1 g, 28%). ¹H NMR (400 MHz,CDCl₃) δ=4.15-4.34 (m, 1H), 1.38-1.63 (m, 4H), 1.16-1.38 (m, 38H),0.93-1.09 (m, 2H), 0.83-0.93 (m, 8H).

Intermediate 69e: 5-octyltridecane-1,3-diol

Intermediate 69d (180 mg, 0.410 mmol) was dissolved in MeOH (2 ml) andTHF (2 ml). 3N NaOH (0.410 ml, 1.231 mmol) was added, followed by 43 uL(0.493 mmol) of a 35% solution of H₂O₂. The reaction was stirred atambient temperature for 2 h. The solvents were removed under reducedpressure, and the crude mixture was purified on silica gel with 0-100%EtOAc/heptanes to provide the title compound as a colorless oil (180mg). ¹H NMR (400 MHz, CDCl₃) δ=3.78-4.02 (m, 3H), 1.60-1.73 (m, 2H),1.42-1.58 (m, 3H), 1.14-1.39 (m, 28H), 0.88 (t, J=6.82 Hz, 6H).

Intermediate 69f: (9Z,12Z)-3-hydroxy-5-octyltridecyloctadeca-9,12-dienoate

Linoleic acid (164 mg, 0.584 mmol), EDC.HCl (112 mg, 0.584 mmol), andDMAP (47.6 mg, 0.390 mmol) were dissolved in 3 ml DCE. DIPEA (0.255 ml,1.461 mmol) was added and the reaction was stirred at ambienttemperature for 30 min. A solution of Intermediate 69e (160 mg, 0.487mmol) in 3 ml DCE was added. The reaction was stirred at ambienttemperature overnight. The crude reaction mixture was purified on silicagel (pre-equilibrated with 1% NH₄OH in MeOH) eluting with 0-15%EtOAc/heptanes to provide the title compound (180 mg, 63%). ¹H NMR (400MHz, CDCl₃) δ=5.16-5.46 (m, 4H), 4.36 (ddd, J=11.18, 8.53, 5.31 Hz, 1H),4.15 (dt, J=11.18, 5.65 Hz, 1H), 3.74 (dd, J=7.58, 4.04 Hz, 1H), 2.77(t, J=6.57 Hz, 2H), 2.25-2.38 (m, 2H), 1.99-2.13 (m, 4H), 1.93-1.99 (m,1H), 1.73-1.87 (m, 1H), 1.56-1.73 (m, 3H), 1.40-1.55 (m, 2H), 1.21-1.40(m, 42H), 0.89 (dq, J=6.82, 3.45 Hz, 9H).

Example 69:(9Z,12Z)-3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-5-octyltridecyloctadeca-9,12-dienoate

Intermediate 69f (180 mg, 0.305 mmol), DMAP (37.2 mg, 0.305 mmol), andpyridine (49.2 μL, 0.609 mmol) were dissolved into DCE (5000 μL).4-nitrophenyl chloroformate (80 mg, 0.396 mmol) was added. The reactionwas stirred at ambient temperature overnight. Then, 500 uL of3-(dimethylamino)propan-1-ol was added and the reaction was stirred for30 min. The crude yellow mixture was purified on silica gel with 0-100%EtOAc/heptanes, then with 0-10% MeOH/DCM. The material was then purifiedon PL-HCO3 MP-SPE columns eluting with 0-50% MeOH/DCM. The solvents wereremoved to provide the title compound (100 mg, 46%). ¹H NMR (400 MHz,CDCl₃) δ=5.24-5.47 (m, 4H), 4.78-4.97 (m, 1H), 4.16 (dt, J=16.86, 6.60Hz, 4H), 2.78 (t, J=6.57 Hz, 2H), 2.39 (br. s., 2H), 2.23-2.35 (m, 8H),2.06 (q, J=6.82 Hz, 4H), 1.79-1.99 (m, 4H), 1.53-1.73 (m, 3H), 1.17-1.46(m, 44H), 0.82-0.98 (m, 9H). ¹³C NMR (101 MHz, CDCl₃) δ=173.37, 154.59,129.90, 129.72, 127.72, 127.59, 73.88, 65.89, 60.22, 55.65, 44.99 (2C),38.41, 33.95, 33.59, 33.31, 33.25, 33.04, 31.59 (2C), 31.20, 29.73,29.71, 29.30 (4C), 29.03 (4C), 28.88, 28.83, 26.88 (2C), 26.02, 25.94,25.31, 24.59, 22.36 (2C), 22.24, 13.78 (2C), 13.74.

Synthesis of Example 70 Intermediate 70a: 3-hydroxy-5-octyltridecyldecanoate

Intermediate 69e (300 mg, 0.913 mmol) and decanoic acid (189 mg, 1.096mmol) were dissolved in DCE (10 ml). EDC.HCl (210 mg, 1.096 mmol) andDMAP (66.9 mg, 0.548 mmol) were added. Lastly, DIPEA (638 μL, 3.65 mmol)was added. The reaction was stirred at ambient temperature overnight.The crude reaction mixture was purified on silica gel with 0-50%EtOAc/heptanes to provide the title compound as a colorless oil (250 mg,57%). ¹H NMR (400 MHz, CDCl₃) δ=4.38 (ddd, J=5.1, 8.5, 11.2 Hz, 1H),4.15 (td, J=5.7, 11.2 Hz, 1H), 3.83-3.65 (m, 1H), 2.37-2.25 (m, 2H),1.81 (dddd, J=3.3, 5.7, 8.7, 14.4 Hz, 2H), 1.73-1.57 (m, 3H), 1.54-1.40(m, 2H), 1.40-1.14 (m, 40H), 0.89 (t, J=6.7 Hz, 9H).

Example 70: 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-5-octyltridecyldecanoate

¹H NMR (400 MHz, CDCl₃) δ=4.80-4.99 (m, 1H) 4.06-4.27 (m, 4H) 2.43 (t,J=7.20 Hz, 2H) 2.21-2.34 (m, 8H) 1.81-2.00 (m, 4H) 1.55-1.71 (m, 3H)1.35-1.51 (m, 2H) 1.12-1.35 (m, 40H) 0.78-0.95 (m, 9H). MS (M+1)=612.8,Rt=0.94 min (LC Method 6).

Synthesis of Example 71 Intermediate 71a:7-octyl-1-((tetrahydro-2H-pyran-2-yl)oxy)pentadecan-5-ol

In an oven dried round bottom flask charged with N₂, were addedmagnesium turnings (0.196 g, 8.07 mmol) and THF (10 mL).2-(4-Bromobutoxy)tetrahydro-2H-pyran (1.595 g, 6.73 mmol) and iodine(0.017 g, 0.067 mmol) were added. The mixture was heated to reflux, thencooled and stirred for 1 h at room temp. The reaction was cooled in anice-water bath, and Intermediate 69b (1.9 g, 6.73 mmol) was added. Thereaction was stirred for 30 min, then the ice bath was removed and thereaction stirred for an additional 1 h. The reaction was quenched withsat. aq. NH₄Cl and then filtered over celite with EtOAc washes. Theorganic layer was collected, and the aqueous layer was extracted oncemore with EtOAc. The combined organics were dried over Na₂SO₄, filtered,and the filtrate was concentrated under reduced pressure. The crudematerial was purified on silica gel with 0-100% EtOAc/heptane to providethe title compound as a colorless oil (1.7 g, 57%). ¹H NMR (400 MHz,CDCl₃) δ=4.52-4.64 (m, 1H), 3.82-3.95 (m, 1H), 3.63-3.82 (m, 2H),3.46-3.57 (m, 1H), 3.34-3.46 (m, 1H), 1.79-1.96 (m, 1H), 1.68-1.79 (m,1H), 1.37-1.67 (m, 11H), 1.14-1.37 (m, 30H), 0.82-0.98 (m, 6H).

Intermediate 71b: 3-(dimethylamino)propyl(7-octyl-1-((tetrahydro-2H-pyran-2-yl)oxy)pentadecan-5-yl) carbonate

p-Nitrophenyl chloroformate (1.244 g, 6.17 mmol) and pyridine (0.936 mL,11.57 mmol) were dissolved in DCM (30 mL). Intermediate 71a (1.7 g, 3.86mmol) in 10 mL of DCM was added via syringe. DMAP (0.236 g, 1.929 mmol)was then added and the mixture was stirred at ambient temperature for 6h, then 3-(dimethylamino)propan-1-ol (0.796 g, 7.71 mmol) was added andthe reaction is stirred overnight. The reaction mixture was purified onsilica gel with 0-100% EtOAc/heptanes, then 0-10% MeOH/DCM to providethe title compound (320 mg, 15%). ¹H NMR (400 MHz, CDCl₃) δ=4.78 (br.s., 1H), 4.64-4.52 (m, 1H), 4.32-4.02 (m, 4H), 3.94-3.80 (m, 1H),3.78-3.67 (m, 1H), 3.61-3.46 (m, 2H), 3.46-3.30 (m, 1H), 2.65-2.44 (m,2H), 2.35 (s, 6H), 1.99-1.88 (m, 2H), 1.72 (d, J=3.0 Hz, 1H), 1.70-1.47(m, 7H), 1.47-1.33 (m, 4H), 1.33-1.08 (m, 28H), 1.07-0.77 (m, 6H).

Intermediate 71c: 3-(dimethylamino)propyl(1-hydroxy-7-octylpentadecan-5-yl) carbonate

1 N HCl (2.91 ml, 2.91 mmol) was added to a solution of Intermediate 71b(553 mg, 0.970 mmol) in MeOH (10 ml) and DCM (1 mL). The reaction wasstirred at ambient temperature for 18 h. The reaction was concentratedunder reduced pressure, then the residue was rediluted with DCM andwashed with aq sodium bicarbonate solution. The aqueous layer wasback-extracted with DCM. The combined organic extracts were dried overMgSO₄, then filtered, and the filtrate was concentrated under reducedpressure to provide the title compound (490 mg), which was used withoutfurther purification. ¹H NMR (400 MHz, CDCl₃) δ=4.74-4.87 (m, 1H),4.10-4.27 (m, 2H), 3.59-3.71 (m, 2H), 2.30-2.45 (m, 2H), 2.23 (s, 6H),1.85 (quin, J=6.95 Hz, 2H), 1.51-1.71 (m, 5H), 1.35-1.51 (m, 4H),1.16-1.35 (m, 28H), 0.80-0.97 (m, 6H).

Example 71:5-(((3-(dimethylamino)propoxy)carbonyl)oxy)-7-octylpentadecyl octanoate

Intermediate 71c (250 mg, 0.515 mmol) and decanoic acid (82 mg, 0.566mmol) were dissolved in DCE (5146 μL). EDC.HCl (118 mg, 0.618 mmol) andDMAP (62.9 mg, 0.515 mmol) were added. Then, DIPEA (270 μL, 1.544 mmol)was added. The reaction was stirred at ambient temperature for 3 h. Thereaction was transferred to a sealed vial and was heated under microwaveirradiation to 80° C. for 20 min. The crude mixture was purified onsilica gel with 0-100% EtOAc/heptanes, then 0-15% MeOH/DCM to providethe title compound (168 mg, 53%). ¹H NMR (400 MHz, CDCl₃) δ=4.69-4.88(m, 1H), 4.13-4.26 (m, 2H), 4.06 (t, J=6.57 Hz, 2H), 2.36 (t, J=7.33 Hz,2H), 2.29 (t, J=7.58 Hz, 2H), 2.23 (s, 6H), 1.85 (quin, J=7.01 Hz, 2H),1.51-1.74 (m, 7H), 1.35-1.50 (m, 4H), 1.15-1.35 (m, 36H), 0.80-0.97 (m,9H). MS (M+1)=612.5, Rt=1.01 min (LC Method 6).

Example 72:(9Z,12Z)-5-(((3-(dimethylamino)propoxy)carbonyl)oxy)-7-octylpentadecyloctadeca-9,12-dienoate

¹H NMR (400 MHz, CDCl₃) δ=5.27-5.46 (m, 4H), 4.73-4.86 (m, 1H),4.12-4.26 (m, 2H), 4.06 (t, J=6.69 Hz, 2H), 2.78 (t, J=6.69 Hz, 2H),2.32-2.42 (m, 2H), 2.29 (t, J=7.58 Hz, 2H), 2.23 (s, 6H), 2.06 (q,J=6.82 Hz, 4H), 1.78-1.91 (m, 2H), 1.51-1.73 (m, 7H), 1.17-1.46 (m,46H), 0.81-0.95 (m, 9H). MS (M+1)=748.6, Rt=1.33 min (LC Method 6).

Example 73:9-(((3-(dimethylamino)propoxy)carbonyl)oxy)-11-octylnonadecyl octanoate

¹H NMR (400 MHz, CDCl₃) δ=4.72-4.85 (m, 1H), 4.18 (t, J=6.44 Hz, 2H),4.06 (t, J=6.69 Hz, 2H), 2.42-2.57 (m, 2H), 2.25-2.37 (m, 8H), 1.85-1.99(m, 2H), 1.49-1.71 (m, 7H), 1.38 (br. s., 3H), 1.16-1.34 (m, 45H),0.84-0.95 (m, 9H). MS (M+1)=668.5, Rt=1.83 min (LC Method 4).

Example 74:9-(((3-(dimethylamino)propoxy)carbonyl)oxy)-11-octylnonadecyl decanoate

¹H NMR (400 MHz, CDCl₃) δ=4.72-4.86 (m, 1H), 4.18 (t, J=6.69 Hz, 2H),4.06 (t, J=6.82 Hz, 2H), 2.37 (t, J=7.45 Hz, 2H), 2.29 (t, J=7.58 Hz,2H), 2.23 (s, 6H), 1.75-1.93 (m, 2H), 1.51-1.70 (m, 7H), 1.34-1.46 (m,4H), 1.16-1.34 (m, 48H), 0.81-0.94 (m, 9H). MS (M+1)=696.3, Rt=2.49 min(LC Method 4).

Example 75:(9Z,12Z)-9-(((3-(dimethylamino)propoxy)carbonyl)oxy)nonadecyloctadeca-9,12-dienoate

¹H NMR (400 MHz, CDCl₃) δ=5.31-5.46 (m, 4H), 4.62-4.76 (m, 1H), 4.19 (t,J=6.57 Hz, 2H), 4.06 (t, J=6.82 Hz, 2H), 2.78 (t, J=6.57 Hz, 2H), 2.39(t, J=7.33 Hz, 2H), 2.29 (t, J=7.45 Hz, 2H), 2.25 (s, 6H), 1.99-2.12 (m,4H), 1.79-1.95 (m, 2H), 1.47-1.71 (m, 8H), 1.19-1.45 (m, 40H), 0.79-0.97(m, 6H). MS (M+1)=692.5, Rt=1.22 min (LC Method 6).

Example 76: 9-(((3-(dimethylamino)propoxy)carbonyl)oxy)nonadecylhexanoate

¹H NMR (400 MHz, CDCl₃) δ=4.57-4.78 (m, 1H), 4.19 (t, J=6.57 Hz, 2H),4.06 (t, J=6.82 Hz, 2H), 2.41 (br. s., 2H), 2.27 (d, J=4.29 Hz, 6H),1.81-1.99 (m, 2H), 1.48-1.69 (m, 8H), 1.20-1.42 (m, 32H), 0.90 (d,J=6.82 Hz, 6H). MS (M+1)=528.3, Rt=1.04 min (LC Method 6).

Example 77: 9-(((3-(dimethylamino)propoxy)carbonyl)oxy)nonadecyl3-octylundecanoate

¹H NMR (400 MHz, CDCl₃) δ=4.60-4.76 (m, 1H), 4.18 (t, J=6.57 Hz, 2H),4.05 (t, J=6.69 Hz, 2H), 2.39 (s, 2H), 2.20-2.31 (m, 8H), 1.75-1.94 (m,3H), 1.43-1.67 (m, 6H), 1.21-1.39 (m, 54H), 0.84-1.05 (m, 9H). MS(M+1)=710.5, Rt=1.33 min (LC Method 6).

Example 78: 9-((4-(dimethylamino)butanoyl)oxy)nonadecyl hexanoate

¹H NMR (400 MHz, CDCl₃) δ=4.87 (t, J=6.19 Hz, 1H), 4.05 (t, J=6.69 Hz,2H), 2.30 (td, J=7.89, 5.68 Hz, 4H), 2.25-2.37 (m, 2H), 2.22 (s, 6H),1.79 (dq, J=7.58, 7.41 Hz, 2H), 1.55-1.70 (m, 4H), 1.50 (d, J=6.06 Hz,4H), 1.17-1.39 (m, 30H), 0.89 (q, J=6.99 Hz, 6H). ¹³C NMR ¹³C NMR (101MHz, CDCl₃) δ=173.97, 173.37, 74.19, 64.34 (2C), 58.95, 45.44 (2C),34.35, 34.13 (2C), 32.47, 31.89, 31.31, 29.57, 29.53 (2C), 29.44, 29.41,29.31, 29.17, 28.63, 25.89, 25.31, 25.30, 24.69, 23.17, 22.66, 22.31,14.09, 13.90.

Example 79: 9-((4-(dimethylamino)butanoyl)oxy)nonadecyl3-octylundecanoate

¹H NMR (400 MHz, CDCl₃) δ=4.86 (t, J=6.19 Hz, 1H), 4.04 (t, J=6.69 Hz,2H), 2.30 (dt, J=16.48, 7.42 Hz, 4H), 2.21 (s, 6H), 2.22 (d, J=6.82 Hz,2H), 1.71-1.88 (m, 3H), 1.59 (d, J=7.33 Hz, 2H), 1.50 (d, J=5.81 Hz,4H), 1.25 (s, 48H), 1.30 (br. s., 6H), 0.88 (d, J=13.64 Hz, 9H). ¹³C NMR(101 MHz, CDCl₃) δ=173.75, 173.35, 74.18, 64.23, 58.93, 45.41 (2C),39.34, 35.08, 34.12, 33.88, 32.45, 31.88 (3C), 29.89 (3C), 29.56 (4C),29.52 (2C), 29.47, 29.43, 29.29 (3C), 29.20, 28.65, 26.51 (2C), 25.95,25.31 (2C), 23.14, 22.65 (4C), 14.08 (3C)

Synthesis of Example 80 Intermediate 80a

Intermediate 80a can be synthesized using methods similar to those usedfor the synthesis of Intermediate 1k. MS (M+1)=387.9, Rt=0.41 min (LCMethod 4).

Intermediate 80b: Synthesis of 3-(dimethylamino)propyl(1-oxohexadecan-4-yl) carbonate

Intermediate 80a was dissolved in DCM (50 ml) and cooled in an ice bath.TEA (7.80 ml, 56.3 mmol) was then added. In another flask, SO₃Py (5.97g, 37.5 mmol) was dissolved in DMSO (17.59 g, 225 mmol), and theresultant solution was added dropwise to the cold DCM solution. Theresultant mixture was stirred at room temperature overnight. Thereaction was then diluted with 800 mL ethyl acetate and the organicphase was washed with water three times. The combined aqueous phase wasback-extracted with 200 mL EtOAc twice. The combined organic extractswere dried over sodium sulfate and filtered. The filtrate wasconcentrated under reduced pressure and purified on silica gel with0-10% MeOH/DCM to afford 4.8 g of the title compound. MS (M+1)=386.6,Rt=1.28 min (LC Method 11).

Intermediate 80c: Synthesis of4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoic acid

In a 500 mL round-bottomed flask Intermediate 80b (5 g, 12.97 mmol) and2-methylbut-2-ene (4.55 gram, 64.8 mmol) were dissolved in t-BuOH(Volume: 25 ml, Ratio: 1.000) and 4N formic acid buffer, pH 3.5 (25 ml).A 2 M aq solution of NaClO₂ (9.73 ml, 19.45 mmol) was added. Thereaction was stirred at ambient temperature for one hour. The mixturewas diluted with 800 mL DCM and 50 mL water. 1 N HCl was added to adjustthe pH of the aqueous layer to 5 The aqueous layer was extracted with 5%MeOH in DCM. The combined organics were dried over Na₂SO₄ and filtered.The filtrate was concentrated under reduced pressure and purified onsilica gel The material was redissolved in 5% MeOH in DCM and washedwith sat. aq. NaHCO₃. The organic phase was dried over Na₂SO₄, filteredand the filtrate concentrated concentrated under reduced pressure toafford 4.5 g of the title compound. MS (M+1)=402.6, Rt=1.23 min (LCMethod 11).

Example 80:(9Z,9′Z,12Z,12′Z)-2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diylbis(octadeca-9,12-dienoate)

In a round bottom flask, Intermediate 80c (4.5 g, 11.21 mmol), DMAP (548mg, 4.48 mmol), and 1,3-Dilinolein (10.37 g, 16.81 mmol) were taken intodichloromethane (100 ml). EDC.HCl (4.3 g, 22.41 mmol) was added in oneportion followed by DIPEA (3.91 ml, 22.41 mmol), dropwise, and thereaction was stirred at ambient temperature. After 24 h, the reactionwas concentrated under reduced pressure. The residue was purified onsilica gel with 0-60% ethyl acetate/heptane to provide the titlecompound (6.17 g). ¹H NMR (400 MHz, CDCl₃) δ=5.19-5.48 (m, 9H),4.65-4.81 (m, 1H), 4.30 (dd, J=11.80, 4.27 Hz, 2H), 4.08-4.25 (m, 4H),2.77 (t, J=6.40 Hz, 4H), 2.36-2.52 (m, 4H), 2.20-2.36 (m, 10H), 2.05 (q,J=6.78 Hz, 8H), 1.81-2.00 (m, 5H), 1.48-1.71 (m, 7H), 1.16-1.44 (m,46H), 0.81-0.96 (m, 9H). MS (M+1)=1001.4, Rt=1.30 min (LC Method 4).

Example 81:(9Z,9′Z,12Z,12′Z)-2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diylbis(octadeca-9,12-dienoate)

¹H NMR (400 MHz, CDCl₃) δ=5.20-5.48 (m, 9H), 4.72 (br. s., 1H), 4.30(dd, J=11.80, 4.27 Hz, 2H), 4.06-4.25 (m, 4H), 2.78 (t, J=6.65 Hz, 4H),2.47-2.60 (m, 6H), 2.37-2.47 (m, 2H), 2.25-2.37 (m, 4H), 2.05 (q, J=6.78Hz, 7H), 1.73-2.01 (m, 4H), 1.48-1.73 (m, 6H), 1.17-1.45 (m, 48H), 1.02(t, J=7.15 Hz, 6H), 0.81-0.95 (m, 9H). MS (M+1)=1029.0, Rt=1.51 min (LCMethod 6).

Example 82:(9Z,9′Z,12Z,12′Z,15Z,15′Z)-2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diylbis(octadeca-9,12,15-trienoate)

¹H NMR (400 MHz, CDCl₃) δ=5.48-5.19 (m, 13H), 4.79-4.63 (m, 1H), 4.30(dd, J=4.1, 11.9 Hz, 2H), 4.24-4.05 (m, 4H), 2.91-2.68 (m, 8H),2.52-2.26 (m, 8H), 2.23 (s, 6H), 2.15-2.01 (m, 8H), 2.01-1.77 (m, 4H),1.70-1.45 (m, 6H), 1.42-1.28 (m, 20H), 1.26 (m, 16H), 0.98 (t, J=7.5 Hz,6H), 0.92-0.83 (m, 3H). ¹³C NMR (101 MHz, CDCl₃) δ=173.26 (2C), 172.05,155.02, 131.94 (2C), 130.22 (2C), 128.26 (2C), 128.21 (2C), 127.70 (2C),127.07 (2C), 77.53, 69.20, 66.32, 61.99, 61.96, 55.96, 45.46 (2C),34.07, 33.96 (2C), 31.90, 30.01, 29.66, 29.63 (2C), 29.57 (2C), 29.50,29.46, 29.35, 29.17 (2C), 29.11 (2C), 29.07 (2C), 28.96, 27.19 (3C),26.95, 25.59 (2C), 25.50 (2C), 25.14, 24.79 (2C), 22.68, 20.53 (2C),14.28 (2C), 14.13.

Example 83:(Z)-2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyldioleate

¹H NMR (400 MHz, CDCl₃) δ=5.44-5.30 (m, 4H), 5.30-5.20 (m, 1H),4.82-4.63 (m, 1H), 4.30 (dd, J=4.4, 11.9 Hz, 2H), 4.25-4.07 (m, 4H),2.50-2.27 (m, 8H), 2.23 (s, 6H), 2.07-1.93 (m, 9H), 1.93-1.76 (m, 3H),1.70-1.48 (m, 6H), 1.42-1.17 (m, 60H), 0.94-0.81 (m, 9H). ¹³C NMR (101MHz, CDCl₃) δ=173.28 (2C), 172.05, 155.02, 129.99 (2C), 129.70 (2C),77.53, 69.20, 66.33, 61.99, 61.95, 55.97, 45.47 (2C), 34.08, 33.97 (2C),31.90 (3C), 30.01, 29.75 (2C), 29.70 (2C), 29.66 (3C), 29.64 (2C),29.57, 29.52 (3C), 29.46, 29.35, 29.32 (5C), 29.18, 29.11, 29.09, 28.97,27.20 (2C), 27.16 (2C), 26.96, 25.15, 24.80 (2C), 22.68 (3C), 14.13(3C).

Example 84:2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diylditetradecanoate

¹H NMR (400 MHz, CDCl₃) δ=5.22-5.30 (m, 1H), 4.67-4.78 (m, 1H), 4.30(dd, J=12.05, 4.27 Hz, 2H), 4.10-4.24 (m, 4H), 2.54 (m, 6H), 2.37-2.48(m, 2H), 2.26-2.37 (m, 4H), 1.78-2.04 (m, 4H), 1.48-1.69 (m, 6H), 1.26(m, 60H), 1.03 (t, J=7.15 Hz, 6H), 0.83-0.93 (m, 9H). ¹³C NMR (101 MHz,CDCl₃): δ ppm 173.3 (s, 2C), 172.1, 155.0, 77.5, 69.2, 66.5, 62.0 (d,2C), 49.0, 46.9 (s, 2C), 34.1, 34.0 (s, 2C), 31.9 (s, 3C), 30.0,29.7-29.3 (overlap, 23), 29.1 (s, 2C), 29.0, 26.3 (br s, 1C), 25.1, 24.8(s, 2C), 22.7 (s, 2C), 14.1 (s, 2C), 11.6 (br s, 2C)

Example 85:2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diylditetradecanoate

¹H NMR (400 MHz, CDCl₃) δ=5.20-5.33 (m, 1H), 4.66-4.79 (m, 1H),4.25-4.37 (m, 2H), 4.08-4.24 (m, 4H), 2.35-2.48 (m, 4H), 2.28-2.35 (m,4H), 2.25 (s, 6H), 1.81-2.02 (m, 4H), 1.49-1.70 (m, 6H), 1.20-1.40 (m,60H), 0.84-0.93 (m, 9H). MS (M+1)=896.9, Rt=1.51 min (LC Method 6).

Example 86:2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diylditetradecanoate

¹H NMR (400 MHz, CDCl₃) δ=5.17-5.35 (m, 1H), 4.65-4.79 (m, 1H), 4.30(dd, J=11.92, 4.39 Hz, 2H), 4.08-4.24 (m, 4H), 2.18-2.78 (m, 13H),1.80-2.06 (m, 4H), 1.47-1.71 (m, 6H), 1.17-1.40 (m, 60H), 1.05-1.17 (m,3H), 0.80-0.96 (m, 9H). MS (M+1)=911.0, Rt=4.54 min (LC Method 4).

Example 87:2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyldidodecanoate

¹H NMR (400 MHz, CDCl₃) δ=5.26 (m, 1H), 4.65-4.80 (m, 1H), 4.30 (dd,J=11.92, 4.39 Hz, 2H), 4.04-4.24 (m, 4H), 2.35-2.49 (m, 4H), 2.28-2.35(m, 4H), 2.25 (s, 6H), 1.93-2.03 (m, 1H), 1.80-1.93 (m, 3H), 1.48-1.70(m, 6H), 1.17-1.45 (m, 52H), 0.83-0.94 (m, 9H). MS (M+1)=840.8, Rt=1.32min (LC Method 3).

Example 88:2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyldidodecanoate

¹H NMR (400 MHz, CDCl₃) δ=5.26 (quin, J=5.08 Hz, 1H) 4.66-4.79 (m, 1H)4.30 (dd, J=11.92, 4.39 Hz, 2H) 4.09-4.24 (m, 4H) 2.54 (m, 6H) 2.36-2.49(m, 2H) 2.24-2.36 (m, 4H) 1.78-2.02 (m, 4H) 1.49-1.69 (m, 6H) 1.18-1.40(m, 52H) 1.03 (t, J=7.03 Hz, 6H) 0.88 (t, J=6.78 Hz, 9H). MS(M+1)=868.9, Rt=1.96 min (LC Method 7).

Example 89:2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyldidodecanoate

¹H NMR (400 MHz, CDCl₃) δ=5.20-5.31 (m, 1H), 4.67-4.78 (m, 1H), 4.30(dd, J=12.05, 4.27 Hz, 2H), 4.06-4.24 (m, 4H), 2.46-2.71 (m, 4H),2.37-2.46 (m, 2H), 2.20-2.37 (m, 7H), 1.79-2.05 (m, 4H), 1.47-1.71 (m,6H), 1.18-1.45 (m, 52H), 1.12 (br. s., 3H), 0.79-0.94 (m, 9H). MS(M+1)=855.0, Rt=3.13 min (LC Method 4).

Example 90:2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diylbis(decanoate)

¹H NMR (400 MHz, CDCl₃) δ=5.21-5.31 (m, 1H), 4.67-4.78 (m, 1H), 4.30(dd, J=12.05, 4.27 Hz, 2H), 4.10-4.25 (m, 4H), 2.48-2.64 (m, 6H),2.36-2.48 (m, 2H), 2.27-2.36 (m, 4H), 1.78-2.02 (m, 4H), 1.48-1.69 (m,6H), 1.19-1.41 (m, 44H), 1.03 (t, J=7.15 Hz, 6H), 0.88 (t, J=6.78 Hz,9H). MS (M+1)=813.0, Rt=1.45 min (LC Method 7).

Example 91:2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diylbis(decanoate)

¹H NMR (400 MHz, CDCl₃) δ=5.26 (quin, J=5.02 Hz, 1H), 4.67-4.78 (m, 1H),4.30 (dd, J=11.80, 4.27 Hz, 2H), 4.08-4.24 (m, 4H), 2.44-2.63 (m, 4H),2.36-2.44 (m, 2H), 2.20-2.36 (m, 7H), 1.79-2.04 (m, 4H), 1.48-1.72 (m,6H), 1.19-1.38 (m, 44H), 1.10 (t, J=6.53 Hz, 3H), 0.80-0.94 (m, 9H). MS(M+1)=798.9, Rt=1.57 min (LC Method 7).

Example 92:2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyldioctanoate

¹H NMR (400 MHz, CDCl₃) δ=5.26 (quin, J=5.0 Hz, 1H), 4.78-4.66 (m, 1H),4.30 (dd, J=4.3, 11.8 Hz, 2H), 4.24-4.10 (m, 4H), 2.64-2.47 (m, 6H),2.47-2.37 (m, 2H), 2.37-2.26 (m, 4H), 2.02-1.78 (m, 4H), 1.70-1.48 (m,6H), 1.39-1.19 (m, 36H), 1.03 (t, J=7.2 Hz, 6H), 0.88 (t, J=6.8 Hz, 9H).MS (M+1)=755.9, Rt=1.89 min (LC Method 7).

Example 93:2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyldioctanoate

¹H NMR (400 MHz, CDCl₃) δ=5.19-5.32 (m, 1H), 4.66-4.79 (m, 1H), 4.30(dd, J=11.92, 4.39 Hz, 2H), 4.05-4.24 (m, 4H), 2.44-2.70 (m, 4H),2.37-2.44 (m, 2H), 2.20-2.37 (m, 7H), 1.80-2.05 (m, 4H), 1.48-1.71 (m,6H), 1.18-1.42 (m, 36H), 1.10 (d, J=6.53 Hz, 3H), 0.76-0.95 (m, 9H). MS(M+1)=742.7, Rt=1.34 min (LC Method 7).

Synthesis of Example 94 Intermediate 94a

Intermediate 94a can be synthesized using methods similar to thoseexemplified for the synthesis of Intermediate 49a. ¹H NMR (400 MHz,CDCl₃) δ=5.26-5.47 (m, 4H), 3.64-3.73 (m, 2H), 3.54-3.64 (m, 1H), 2.78(t, J=6.40 Hz, 2H), 2.06 (q, J=6.94 Hz, 4H), 1.56-1.74 (m, 3H),1.40-1.52 (m, 4H), 1.16-1.40 (m, 18H), 0.91 (s, 9H), 0.86-0.90 (m, 3H),0.08 (s, 6H).

Example 94:2-(((13Z,16Z)-4-(((3-(dimethylamino)propoxy)carbonyl)oxy)docosa-13,16-dienoyl)oxy)propane-1,3-diyldioctanoate

Example 94 can be synthesized using methods similar to those exemplifiedfor the synthesis of Example 80. ¹H NMR (400 MHz, CDCl₃) δ=5.45-5.21 (m,5H), 4.73 (d, J=4.3 Hz, 1H), 4.30 (dd, J=4.3, 11.8 Hz, 2H), 4.23-4.07(m, 4H), 2.77 (t, J=6.3 Hz, 2H), 2.49-2.36 (m, 4H), 2.36-2.21 (m, 9H),2.12-1.80 (m, 7H), 1.70-1.48 (m, 6H), 1.40-1.20 (m, 36H), 0.94-0.81 (m,9H) □ppm. LC-MS m/z=808.5 (M+1). Rt=1.59 min (LC Method 7).

Example 95:2-(((13Z,16Z)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)docosa-13,16-dienoyl)oxy)propane-1,3-diyldioctanoate

¹H NMR (CDCl₃) δ=5.30-5.46 (m, 4H), 5.21-5.30 (m, 1H), 4.73 (ddd, J=7.2,4.2, 2.8 Hz, 1H), 4.30 (dd, J=12.0, 4.4 Hz, 2H), 4.10-4.26 (m, 4H), 2.78(t, J=6.4 Hz, 2H), 2.55 (d, J=5.1 Hz, 6H), 2.36-2.47 (m, 2H), 2.27-2.36(m, 4H), 2.02-2.12 (m, 4H), 1.76-2.02 (m, 4H), 1.48-1.72 (m, 6H),1.21-1.42 (m, 34H), 1.04 (t, J=6.9 Hz, 6H), 0.89 (dq, J=6.9, 3.4 Hz,9H). MS (M+1)=836.6, Rt=1.01 min (LC Method 6).

Synthesis of Example 96 Intermediate 96a:2-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecanoic acid

To a solution of Intermediate 48c (2 g, 5.16 mmol), dissolved in acetone(20 mL), cooled in an ice-water bath, was added Jones reagent (5.16 mL,10.32 mmol, 2 M solution in H2O), dropwise. The cooling bath wasremoved, and stirring was continued overnight. iPrOH (1 mL) was added,the mixture was filtered, and the filtrate was removed under reducedpressure. The residue was diluted with water and heptane. The heptanelayer was separated and discarded, and the aqeuous phase was washed withEtOAc, which was also discarded. The pH of the aqueous layer wasadjusted to 6 and the mixture was extracted with 5% MeOH/DCM (5×40 mL).The combined dichloromethane extracts were dried over sodium sulfate andfiltered. The filtrate was concentrated under reduced pressure to afford1 g of the title compound, which was used without further purification.¹H NMR (400 MHz, CDCl₃) δ=4.67-4.78 (m, 1H), 4.23-4.41 (m, 1H), 4.16(dt, J=10.98, 5.43 Hz, 1H), 2.97-3.27 (m, 4H), 2.89 (br. s., 1H),1.98-2.15 (m, 2H), 1.75-1.98 (m, 2H), 1.55-1.75 (m, 1H), 1.39-1.55 (m,3H), 1.09-1.39 (m, 24H), 0.89 (t, J=6.53 Hz, 3H)

Example 96:(9Z,9′Z,12Z,12′Z)-2-((2-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecanoyl)oxy)propane-1,3-diylbis(octadeca-9,12-dienoate)

¹H NMR (400 MHz, CDCl₃) δ=5.28-5.45 (m, 9H), 4.89 (t, J=6.27 Hz, 1H),4.25-4.35 (m, 2H), 4.10-4.25 (m, 4H), 2.77 (t, J=6.53 Hz, 4H), 2.56 (br.s., 6H), 2.26-2.37 (m, 4H), 2.05 (q, J=6.78 Hz, 8H), 1.78-1.95 (m, 3H),1.61 (m, 5H), 1.19-1.50 (m, 48H), 1.06 (br. s., 6H), 0.84-0.94 (m, 9H).MS (M+1)=1000.9, Rt=1.43 min (LC Method 4).

Example 97:(9Z,9′Z,12Z,12′Z)-2-((2-(((3-(dimethylamino)propoxy)carbonyl)oxy)dodecanoyl)oxy)propane-1,3-diylbis(octadeca-9,12-dienoate)

¹H NMR (400 MHz, CDCl₃) δ=5.27-5.45 (m, 9H), 4.89 (t, J=6.27 Hz, 1H),4.25-4.35 (m, 2H), 4.10-4.25 (m, 4H), 2.77 (t, J=6.53 Hz, 4H), 2.37-2.48(m, 2H), 2.22-2.37 (m, 10H), 2.05 (q, J=6.69 Hz, 8H), 1.79-1.95 (m, 5H),1.61 (br. s., 5H), 1.21-1.49 (m, 42H), 0.84-0.95 (m, 9H). ¹³C NMR (101MHz, CDCl₃) δ ppm 173.28, 173.17, 169.42, 154.63, 130.25 (s, 2C), 130.03(d, 2C), 128.07 (s, 2C), 127.90 (s, 2C), 75.13, 70.14, 66.88, 61.90,61.83, 55.85, 45.33 (s, 2C), 33.96, 33.93, 31.93, 31.54, 31.13,29.65-29.13 (overlap, 16C), 27.22 (s, 4C), 26.70, 25.64 (s, 2C), 24.98,24.80, 24.77, 22.71, 22.60 (s, 2C), 14.16, 14.12 (s, 2C).

Example 98:(9Z,9′Z,12Z,12′Z)-2-((2-(((3-(dimethylamino)propoxy)carbonyl)oxy)tetradecanoyl)oxy)propane-1,3-diylbis(octadeca-9,12-dienoate)

¹H NMR (400 MHz, CDCl₃) δ=5.27-5.46 (m, 9H), 4.89 (t, J=6.27 Hz, 1H),4.29 (ddd, J=12.30, 8.66, 4.14 Hz, 2H), 4.09-4.25 (m, 4H), 2.77 (t,J=6.53 Hz, 4H), 2.44 (br. s., 2H), 2.23-2.37 (m, 10H), 2.05 (q, J=6.78Hz, 8H), 1.78-1.98 (m, 4H), 1.61 (br. s., 4H), 1.23-1.50 (m, 48H),0.83-0.96 (m, 9H). MS (M+1)=972.8, Rt=4.25 min (LC Method 4).

Example 99:(9Z,9′Z,12Z,12′Z)-2-((2-(((3-(diethylamino)propoxy)carbonyl)oxy)dodecanoyl)oxy)propane-1,3-diylbis(octadeca-9,12-dienoate)

¹H NMR (400 MHz, CDCl₃) δ=5.31-5.41 (m, 1H), 4.89 (t, J=6.15 Hz, 1H),4.25-4.36 (m, 2H), 4.09-4.25 (m, 4H), 2.59 (br. s., 6H), 2.24-2.37 (m,4H), 1.78-2.03 (m, 4H), 1.61 (d, J=3.26 Hz, 4H), 1.43 (br. s., 2H),1.18-1.37 (d, 34H), 1.07 (br. s., 6H), 0.88 (t, J=6.65 Hz, 9H). MS(M+1)=728.5, Rt=1.37 min (LC Method 7).

Example 100:2-((2-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecanoyl)oxy)propane-1,3-diyldioctanoate)

¹H NMR (400 MHz, CDCl₃) δ=5.31-5.41 (m, 1H), 4.89 (t, J=6.15 Hz, 1H),4.25-4.36 (m, 2H), 4.09-4.25 (m, 4H), 2.59 (br. s., 6H), 2.24-2.37 (m,4H), 1.78-2.03 (m, 4H), 1.61 (d, J=3.26 Hz, 4H), 1.43 (br. s., 2H),1.18-1.37 (d, 34H), 1.07 (br. s., 6H), 0.88 (t, J=6.65 Hz, 9H). MS(M+1)=728.5, Rt=1.37 min (LC Method 7).

Synthesis of Example 101 Intermediate 101a

A 1.0 M solution of DIBAL (15.4 mL, 15.4 mmol) in toluene was added to aDCM (60 mL) solution of Intermediate 11a (5 g, 15.4 mmol), cooled in adry-ice/acetone bath. After 1.5 h, the mixture was allowed to warm tort, and was then treated with sat. aq. ammonium chloride solution (20mL) and water (10 mL). The reaction was extracted with DCM. The combineddichloromethane extracts were washed with brine twice, dried over MgSO₄and filtered. The filtrate was concentrated under reduced pressure andthe residue was dissolved in MeOH (20 mL). NaBH₄ (581 mg, 15.4 mmol) wasadded and the mixture was stirred at rt. After 1 hour, water was addedto the mixture and MeOH was removed under reduced pressure. Theresulting mixture was extracted with EtOAc. The organic extracts werewashed with sat. aq. NaHCO₃, dried over Na₂SO₄ and filtered. Thefiltrate was concentrated under reduced pressure and the residue waspurified on silica gel with 0-20% EtOAc/heptane to afford 2.46 g of thetitle compound. ¹H NMR (400 MHz, DMSO-d₆) δ=4.35-4.45 (m, 2H), 3.47 (dt,J=9.41, 6.46 Hz, 2H), 3.28-3.40 (m, 4H), 1.35-1.57 (m, 8H), 1.24 (br.s., 20H), 0.79-0.91 (m, 6H).

Example 101: 4,4-bis(octyloxy)butyl4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoate

¹H NMR (400 MHz, CDCl₃) δ=4.67-4.77 (m, 1H), 4.44-4.52 (m, 1H),4.13-4.25 (m, 2H), 4.09 (t, J=5.90 Hz, 2H), 3.56 (dt, J=9.16, 6.71 Hz,2H), 3.41 (dt, J=9.22, 6.68 Hz, 2H), 2.30-2.47 (m, 4H), 2.25 (s, 6H),1.81-2.04 (m, 6H), 1.49-1.75 (m, 10H), 1.21-1.40 (m, 38H), 0.82-0.96 (m,9H). MS (M+1)=714.7, Rt=1.83 min (LC Method 7).

Example 102: 4,4-bis(octyloxy)butyl2-(((3-(diethylamino)propoxy)carbonyl)oxy)dodecanoate

¹H NMR (400 MHz, CDCl₃) δ=4.89 (t, J=6.27 Hz, 1H), 4.47 (t, J=5.27 Hz,1H), 4.11-4.28 (m, 4H), 3.56 (dt, J=9.03, 6.78 Hz, 2H), 3.40 (dt,J=9.22, 6.68 Hz, 2H), 2.57 (br. s., 6H), 1.79-1.98 (m, 4H), 1.62-1.77(m, 4H), 1.56 (quin, J=6.84 Hz, 4H), 1.20-1.48 (m, 36H), 1.05 (t, J=6.27Hz, 6H), 0.83-0.94 (m, 9H). MS (M+1)=686.6, Rt=1.57 min (LC Method 7).

Synthesis of Example 103 Intermediate 103a:4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexadecanoic acid

Intermediate 103a can be synthesized using methods similar to thoseexemplified in the synthesis of Intermediate 80c. MS (M+1)=430.2,Rt=0.73 min (LC Method 7).

Intermediate 103b: 1-(bis(2-hydroxyethyl)amino)-1-oxohexadecan-4-yl(3-(diethylamino)propyl) carbonate

In a 100 ml round-bottom flask equipped with a stirbar, Intermediate103a (465 mg, 1.082 mmol) and HATU (453 mg, 1.191 mmol) are dissolved inDCM (Volume: 20 ml). DIPEA (0.756 ml, 4.33 mmol) is added, and mixturestirred for ˜30 min before addition of Diethanolamine (0.311 ml, 3.25mmol). Mixture is then stirred at rt overnite when LCMS shows productformation. The volatiles were then evaporated under pressure, and thecrude material purified on silica gel with 0-20% MeOH/DCM to afford 345mg (62%) of the title compound. LC-MS m/z=623.3 (MH+). Rt=0.43 min (LCMethod 5).

Example 103:(9Z,12Z)-10-dodecyl-3-ethyl-14-(2-((9Z,12Z)-octadeca-9,12-dienoyloxy)ethyl)-8,13-dioxo-7,9-dioxa-3,14-diazahexadecan-16-yloctadeca-9,12-dienoate

In a 500 ml round-bottom flask equipped with a stirbar, Linoleic acid(393 mg, 1.402 mmol) and Intermediate 103b (345 mg, 0.668 mmol) aredissolved in DCM (Volume: 20 ml). DIPEA (0.466 ml, 2.67 mmol) is added,followed by DMAP (32.6 mg, 0.267 mmol) and mixture stirred at rt for ˜5min before addition of EDC.HCl (282 mg, 1.469 mmol). Mixture stirred atrt overnight. The volatiles were then evaporated under reduced pressure,and the crude material purified on silica gel with (0-60% EtOAc/Heptane)to afford 389 mg (53%) of the title compound. ¹H NMR (400 MHz, CDCl₃)δ=5.46-5.27 (m, 8H), 4.82-4.66 (m, 1H), 4.30-4.08 (m, 6H), 3.68-3.52 (m,4H), 2.78 (t, J=6.5 Hz, 4H), 2.70-2.35 (m, 8H), 2.30 (dt, J=3.3, 7.5 Hz,4H), 2.12-1.97 (m, 9H), 1.97-1.78 (m, 3H), 1.71-1.51 (m, 8H), 1.44-1.21(m, 46H), 1.05 (br. s., 6H), 0.95-0.82 (m, 9H). ¹³C NMR (100 MHz, CDCl₃)δ=173.6, 173.4, 172.4, 155.1, 130.2 (2C), 130.0 (2C), 128.0 (2C), 127.9(2C), 78.3, 66.3, 62.0, 61.6, 49.0, 47.1, 46.8, 45.5 (2C), 34.3, 34.1,34.0, 31.9, 31.5 (2C), 29.7 (2C), 29.6 (6C), 29.5 (4C), 29.3 (4C), 29.2(2C), 29.1 (2C), 28.7, 27.2 (4C), 25.6, 25.2, 24.8 (2C), 22.7, 22.6(2C), 14.1 (3C), 11.6 (2C) ppm.

Synthesis of Example 104 Intermediate 104a: tert-butyl 4-oxobutanoate

Intermediate 104a was prepared from tert-butyl 4-hydroxybutanoate in amethod similar to that used for the synthesis of Intermediate 1g. ¹H NMR(CDCl₃) δ=9.83 (s, 1H), 2.73-2.78 (m, 2H), 2.55-2.60 (m, 2H), 1.47 (s,9H)

Intermediate 104b tert-butyl 4-hydroxydodec-11-enoate

Magnesium turnings (307 mg, 12.64 mmol) was weighed into a pre-driedflask and dried in an oven at 120° C. for 2 hours. The flask was removedfrom the oven, sealed, and cooled to ambient temperature. To the flask,6 mL of anhydrous THF and one particle of iodine was added, followed byaddition of 8-bromooct-1-ene (1.570 g, 8.22 mmol). A condenser was addedto the flask, and the whole system was exchanged with N2 and protectedunder N2 ballon. The reaction was heated with a heating until the browncolor from the iodine dissipated. The reaction was heated at refluxuntil most of magnesium was consumed (about 1.5 hr), then cooled to roomtemperature.

To a solution of Intermediate 104a (1.0 g, 6.32 mmol) in 25 ml THFcooled in a dry-ice acetone bath, the freshly prepared Grignard reagentwas added dropwise. The reaction was stirred for 1 h, then warmed toambient temperature. The reaction was quenched with sat. aq. NaHCO₃, andextracted with ethyl acetate. The combined organics were dried oversodium sulfate and filtered. The filtrate was concentrated under reducedpressure and purified on silica gel with 10% ethyl acetate/heptane toafford the desired product (890 mg, 60%). ¹H NMR (400 MHz, CDCl₃)δ=5.61-5.85 (m, 1H), 4.80-4.98 (m, 2H), 3.48-3.62 (m, 1H), 2.26-2.36 (t,J=7.5 Hz, 2H), 1.50-1.78 (m, 4H), 1.35-1.44 (m, 19H).

Intermediate 104c: tert-butyl 4-hydroxy-11-oxoundecanoate

To a solution of Intermediate 104b (300 mg, 0.78 mmol) in 8 mldioxane/H2O (3:1), 2, 6-lutidine (0.36 ml, 3.11 mmol) was added,followed by OsO₄ (0.243 ml 2.5 wt. % in t-butanol, 0.02 mmol), and NalO₄(664 mg, 3.11 mmol). The slurry was stirred for 1.5 h at roomtemperature. The reaction was filtered through celite with ethyl acetatewashes. The filtrate was then washed with brine, dried over sodiumsulfate and filtered. The filtrate was concentrated under reducedpressure and purified on silica gel with 25% ethyl acetate/heptane toafford the desired product (150 mg, 71%). ¹H NMR (400 MHz, CDCl₃) δ=9.76(t, J=1.8 Hz, 1H), 3.51-3.72 (m, 1H), 2.43 (td, J=7.3, 1.9 Hz, 2H), 2.37(t, J=7.2 Hz, 2H), 1.77 (s, 1H), 1.58-1.71 (m, 3H), 1.45 (s, 13H), 1.33(m, 4H).

Intermediate 104d: tert-butyl 4,11-dihydroxyundecanoate

Intermediate 104c (140 mg, 0.51 mmol) was dissolved in 6 ml THF/MeOH(1:1) and cooled in an ice-water bath. NaBH₄ (29.2 mg, 0.77 mmol) wasadded and the mixture was stirred for 30 min. The reaction was warmed toambient temperature then diluted with brine and ethyl acetate. Theorganic extracts were dried over sodium sulfate and filtered. Thefiltrate was concentrated under reduced pressure to provide the titlecompound, which was used in the next step without further purification.¹H NMR (400 MHz, CDCl₃) δ=3.57 (t, J=6.6 Hz, 3H), 2.30 (t, J=7.2 Hz,2H), 1.45-1.77 (m, 7H), 1.33-1.42 (m, 14H), 1.22-130 (m, 4H).

Intermediate 104e: tert-butyl 4-hydroxy-11-(octanoyloxy)undecanoate

To a solution of Intermediate 104d (130 mg, 0.47 mmol) in 4.0 ml DCM,were added octanoic acid (75 mg, 0.52 mmol), DMAP (17.4 mg. 0.14 mmol),DIPEA (0.083 ml, 0.47 mmol), followed by EDC (118 mg, 0.62 mmol). Thereaction was stirred at room temperature for 2 h. The reaction wasdiluted with brine and ethyl acetate. The organic extracts were driedover sodium sulfate and filtered. The filtrate was concentrated underreduced pressure and the residue was purified on silica gel with 10%ethylacetate/heptane to afford the desired product (110 mg, 58%). ¹H NMR(400 MHz, CDCl₃) δ=4.07 (t, J=6.7 Hz, 2H), 3.56-3.68 (m, 1H), 2.38 (t,J=7.3 Hz, 2H), 2.30 (t, J=7.6 Hz, 2H), 1.85 (m, 2H), 1.55-1.74 (m, 5H),1.46 (m, 12H), 1.23-1.41 (m, 15H), 0.82-0.96 (m, 3H).

Intermediate 104f: tert-butyl4-(((3-(diethylamino)propoxy)carbonyl)oxy)-11-(octanoyloxy)undecanoate

To a solution of Intermediate 104e (110 mg, 0.28 mmol) in 3.0 ml DCM,cooled in an ice-water bath, pyridine (0.033 ml, 0.41 mmol) was added,followed by triphosgene (40.7 mg, 0.14 mmol). After 30 min.,3-(diethylamino)-1-propanol (0.126 ml, 0.824 mmol) was added. Theresulted mixture was stirred at room temperature for 1 h. The reactionwas concentrated under reduced pressure, and the residue was purified onsilica gel with 5% MeOH/DCM to afford the desired product (110 mg, 72%).¹H NMR (400 MHz, CDCl₃) δ=4.64-4.88 (m, 1H), 4.24 (t, J=6.8 Hz, 2H),4.06 (t, J=6.8 Hz, 2H), 3.10 (br. s., 6H), 2.18-2.43 (m, 6H), 1.76-2.00(m, 2H), 1.50-1.72 (m, 6H), 1.46 (s, 9H), 1.22-1.45 (m, 22H), 0.86-0.94(m, 3H).

Intermediate 104g:4-(((3-(diethylamino)propoxy)carbonyl)oxy)-11-(octanoyloxy)undecanoicacid

To a solution of Intermediate 104f (110 mg, 0.2 mmol) in 2.4 ml DCM, 0.6ml TFA was added. The resulted mixture stirred at room temperature for 2h. The reaction was dried under reduced pressure to provide the titlecompound, which was used in the next step without further purification.¹H NMR (400 MHz, CDCl₃) δ=10.13-10.42 (br, s, 1H), 5.75 (br. s., 6H),4.71-4.95 (m, 1H), 4.32-4.60 (m, 1H), 4.07 (t, J=6.8 Hz, 3H), 3.08-3.52(m, 6H), 2.47-2.64 (m, 1H), 2.37-2.47 (m, 1H), 2.32 (t, J=7.5 Hz, 2H),2.10 (br. s., 3H), 1.85-2.02 (m, 1H), 1.19-1.44 (m, 22H), 0.80-0.99 (m,3H).

Example 104:2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)-11-(octanoyloxy)undecanoyl)oxy)propane-1,3-diyldioctanoate

¹H NMR (400 MHz, CDCl₃) δ=5.20-5.32 (m, 1H), 4.66-4.79 (m, 1H), 4.29(dd, J=11.8, 4.3 Hz, 2H), 4.09-4.23 (m, 4H), 4.04 (t, J=6.7 Hz, 2H),2.48-2.58 (m, 6H), 2.36-2.48 (m, 2H), 2.24-2.36 (m, 6H), 1.76-2.02 (m,4H), 1.47-1.69 (m, 10H), 1.17-1.41 (m, 32H), 1.02 (t, J=7.2 Hz, 6H),0.81-0.92 (m, 9H). MS (M+1)=828.61, Rt=0.89 min. (LC Method 6).

Synthesis of Example 105 Intermediate 105a:(9Z,9′Z,12Z,12′Z)-2-(hydroxymethyl)propane-1,3-diylbis(octadeca-9,12-dienoate)

In a round bottom flask, linoleic acid (95.0 g, 339 mmol), DMAP (4.14 g,33.90 mmol), DIPEA (74.1 ml, 424 mmol), and2-(hydroxymethyl)propane-1,3-diol (18.0 g, 170 mmol) were taken intodichloromethane (435 ml). EDC (81.0 g, 424 mmol) was added in oneportion, and the reaction was stirred at ambient temperature. After 24h, the reaction is concentrated under reduced pressure with silica gelpowder for dry loading and the residue was purified on silica gel(Biotage) using ethyl acetate/heptane (0% to 40%) as eluent, to provide47 g (44% yield) of the desired product as a colorless oil. ¹H NMR (400MHz, CDCl₃): δ=5.19-5.50 (m, 8H), 4.19 (tt, J=11.83, 5.87 Hz, 4H),3.51-3.69 (m, 2H), 2.78 (t, J=6.53 Hz, 4H), 2.33 (t, J=7.53 Hz, 4H),2.20 (quint, J=5.83 Hz, 2H), 2.06 (q, J=6.78 Hz, 8H), 1.49-1.72 (m, 5H),1.20-1.46 (m, 26H), 0.79-0.98 (m, 6H) ppm.

Example 105:(9Z,9′Z,12Z,12′Z)-2-(9-dodecyl-2-methyl-7,12-dioxo-6,8,13-trioxa-2-azatetradecan-14-yl)propane-1,3-diylbis(octadeca-9,12-dienoate)

Example 105 can be prepared using similar methods to those used tosynthesize Example 80. 1H NMR (400 MHz, CDCl₃): δ=5.55-5.18 (m, 8H),4.71 (dq, J=6.9, 2.7 Hz, 1H), 4.33-4.01 (m, 8H), 2.77 (t, J=6.5 Hz, 4H),2.60-2.17 (m, 15H), 2.17-1.76 (m, 12H), 1.75-1.46 (m, 7H), 1.45-1.16 (m,47H), 0.88 (td, J=6.8, 3.9 Hz, 9H). MS (M+1): 1015.3, Rt=1.28 min (LCMethod XX)

The following example can be prepared using similar sequences andmethods to those employed for the synthesis of Example 11.

Example 106 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl4,4-bis(octyloxy)butanoate

¹H NMR (400 MHz, CD₂Cl₂) δ=4.72-4.82 (m, 1H), 4.45 (t, J=5.56 Hz, 1H),4.03-4.19 (m, 4H), 3.54 (dt, J=9.23, 6.63 Hz, 2H), 3.38 (dt, J=9.20,6.65 Hz, 2H), 2.26-2.39 (m, 4H), 2.19 (s, 6H), 1.75-1.93 (m, 6H),1.45-1.69 (m, 8H), 1.21-1.38 (m, 38H), 0.82-0.93 (m, 9H) ppm. MS(M+1)=701.1, Rt=1.12 min (LC method 12).

The following examples can be prepared using similar sequences andmethods to those employed for the synthesis of Example 16.

Example 107 3-(((3-(piperidin-1-yl)propoxy)carbonyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate

¹H NMR (400 MHz, CD₂Cl₂) δ=4.77 (quin, J=6.24 Hz, 1H), 4.41 (t, J=5.69Hz, 1H), 4.05-4.18 (m, 4H), 3.53 (dt, J=9.29, 6.66 Hz, 2H), 3.37 (dt,J=9.29, 6.66 Hz, 2H), 2.23-2.40 (m, 8H), 1.89 (q, J=6.36 Hz, 2H), 1.80(quin, J=6.88 Hz, 2H), 1.46-1.66 (m, 14H), 1.16-1.45 (m, 44H), 0.83-0.93(m, 9H) ppm.

MS (M+1)=768.8, Rt=2.60 min (LC method 13).

Example 108 3-(((3-(piperazin-1-yl)propoxy)carbonyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate

¹H NMR (400 MHz, CD₂Cl₂) δ=4.77 (quin, J=6.24 Hz, 1H), 4.41 (t, J=5.62Hz, 1H), 4.05-4.18 (m, 4H), 3.53 (dt, J=9.29, 6.66 Hz, 2H), 3.37 (dt,J=9.35, 6.63 Hz, 2H), 2.86 (t, J=4.83 Hz, 4H), 2.34-2.64 (m, 7H), 2.29(t, J=7.52 Hz, 2H), 1.90 (m, J=6.44, 6.44, 6.44 Hz, 2H), 1.81 (quin,J=6.91 Hz, 2H), 1.47-1.66 (m, 10H), 1.21-1.41 (m, 42H), 0.83-0.93 (m,9H) ppm.

MS (M+1)=770.2, Rt=1.18 min (LC method 14).

Example 109 3-(((4-(diethylamino) butoxy)carbonyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate

¹H NMR (400 MHz, CD₂Cl₂) δ=4.77 (quin, J=6.24 Hz, 1H), 4.41 (t, J=5.62Hz, 1H), 4.02-4.20 (m, 4H), 3.53 (dt, J=9.29, 6.66 Hz, 2H), 3.37 (dt,J=9.29, 6.66 Hz, 2H), 2.35-2.56 (m, 6H), 2.28 (t, J=7.52 Hz, 2H), 1.90(q, J=6.40 Hz, 2H), 1.42-1.73 (m, 14H) 1.21-1.40 (m, 42H) 0.98 (t,J=7.09 Hz, 6H) 0.84-0.92 (m, 9H) ppm.

MS (M+1)=771.2, Rt=2.82 min (LC method 13).

Example 1103-(((3-(4-methylpiperazin-1-yl)propoxy)carbonyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate

¹H NMR (400 MHz, CD₂Cl₂) δ=4.77 (quin, J=6.24 Hz, 1H), 4.41 (t, J=5.62Hz, 1H), 4.04-4.19 (m, 4H), 3.53 (dt, J=9.29, 6.66 Hz, 2H), 3.37 (dt,J=9.35, 6.63 Hz, 2H), 2.24-2.55 (m, 12H), 2.22 (s, 3H), 1.89 (q, J=6.36Hz, 2H) 1.80 (quin, J=6.91 Hz, 2H), 1.48-1.66 (m, 10H), 1.19-1.42 (m,42H), 0.81-0.95 (m, 9H) ppm.

MS (M+1)=784.2, Rt=2.64 min (LC method 13).

Example 111 3-((((1-methylpiperidin-4-yl)methoxy)carbonyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate

¹H NMR (400 MHz, ACETONITRILE-d₃): δ=4.76 (quin, J=6.27 Hz, 1H), 4.41(t, J=5.62 Hz, 1H), 4.07 (t, J=6.30 Hz, 2H), 4.01-3.88 (m, 2H), 3.52(dt, J=9.38, 6.56 Hz, 2H), 3.43-3.33 (m, 2H), 2.82-2.72 (m, 2H), 2.26(t, J=7.34 Hz, 3H), 1.90-1.80 (m, 2H) 2.16 (s, 3H), 1.68-1.46 (m, 12H),1.38-1.20 (m, 46H), 0.88 (t, J=6.54 Hz, 9H) ppm.

MS (M+1)=755, Rt=1.20 min (LC method 12).

Example 112 3-(((3-morpholinopropoxy)carbonyl)oxy) pentadecyl6,6-bis(octyloxy) hexanoate

¹H NMR (400 MHz, CDCl₃) δ=4.85-4.76 (m, 1H), 4.45 (t, J=5.69 Hz, 1H),4.26-4.04 (m, 4H), 3.75 (br. s., 4H), 3.56 (dt, J=9.26, 6.68 Hz, 2H),3.40 (dt, J=9.29, 6.72 Hz, 2H), 2.54-2.39 (m, 4H), 2.35-2.26 (m, 2H),1.96-1.88 (m, 2H), 1.70-1.51 (m, 12H), 1.37-1.22 (m, 44H), 0.93-0.85 (m,9H) ppm.

MS (M+1)=771, Rt=1.34 min (LC method 12).

Example 113 3-(((2-(diethylamino)ethoxy)carbonyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate

¹H NMR (400 MHz, CDCl₃) δ=4.86-4.75 (m, 1H), 4.46 (t, J=5.69 Hz, 1H),4.37-4.23 (m, 1H), 4.21-4.06 (m, 2H), 3.56 (dt, J=9.29, 6.66 Hz, 2H),3.40 (dt, J=9.29, 6.72 Hz, 2H), 2.31 (t, J=7.58 Hz, 2H), 1.97-1.87 (m,2H), 1.72-1.49 (m, 12H), 1.44-1.05 (m, 53H), 0.93-0.85 (m, 9H) ppm.

MS (M+1)=743, Rt=0.86 min (LC method 12).

Synthesis of example 114

The following examples can be prepared using similar sequences andmethods to those employed for the synthesis of example 16, coupling withintermediate 114a, derived from 2-propyl penanol.

Intermediate 114a: 6,6-bis((2-propylpentyl)oxy)hexanoic acid

TLC (silica gel, 10% MeOH/DCM, PMA stain): R_(f)=0.14.

Example 114 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl6,6-bis((2-propylpentyl)oxy)hexanoate

¹H NMR (400 MHz, CD₂Cl₂) δ=4.77 (quin, J=6.27 Hz, 1H), 4.37 (t, J=5.62Hz, 1H), 4.02-4.19 (m, 4H), 3.44 (dd, J=9.23, 5.69 Hz, 2H), 3.25 (dd,J=9.23, 5.69 Hz, 2H), 2.48 (q, J=7.09 Hz, 6H), 2.28 (t, J=7.58 Hz, 2H),1.89 (q, J=6.40 Hz, 2H), 1.76 (t, J=6.85 Hz, 2H), 1.47-1.66 (m, 8H),1.16-1.41 (m, 38H), 0.98 (t, J=7.09 Hz, 6H), 0.85-0.92 (m, 15H) ppm.

MS (M+1)=757.2, Rt=2.62 min (LC method 13).

Example 115 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl6,6-bis((2-propylpentyl)oxy)hexanoate

¹H NMR (400 MHz, ACETONITRILE-d₃) δ=4.80-4.71 (m, 1H), 4.38 (t, J=5.62Hz, 1H), 4.17-4.02 (m, 4H), 3.45 (dd, J=9.29, 5.62 Hz, 2H), 3.27 (dd,J=9.29, 5.62 Hz, 2H), 2.27 (td, J=7.21, 4.65 Hz, 4H), 2.14 (s, 6H),1.90-1.82 (m, 2H), 1.76 (quin, J=6.85 Hz, 2H), 1.64-1.47 (m, 8H),1.40-1.19 (m, 38H), 0.88 (t, J=7.03 Hz, 15H) ppm.

MS (M+1)=729, Rt=1.16 min (LC method 14).

Synthesis of Example 116

The following example can be prepared using similar sequences andmethods to those employed for the synthesis of example 16, but withcoupling partner intermediate 116a, derived from 3-ethyl-1-pentanol.

Intermediate 116a: 6,6-bis((3-ethylpentyl)oxy)hexanoic acid

TLC (silica gel, 10% MeOH/DCM, PMA stain): R_(f)=0.17.

Example 116 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl6,6-bis((3-ethylpentyl)oxy)hexanoate

¹H NMR (400 MHz, CD₂Cl₂) δ: 4.82-4.72 (m, 1H), 4.41 (t, J=5.6 Hz, 1H),4.19-4.03 (m, 4H), 3.56 (dt, J=9.3, 7.1 Hz, 2H), 3.40 (dt, J=9.3, 7.1Hz, 2H), 2.30 (dt, J=10.3, 7.4 Hz, 4H), 2.18 (s, 6H), 1.90 (q, J=6.4 Hz,2H), 1.84-1.74 (m, 2H), 1.67-1.53 (m, 6H), 1.53-1.45 (m, 4H), 1.41-1.19(m, 32H), 0.91-0.79 (m, 15H).

MS (M+1)=700.6, Rt=2.34 min (LC method 13).

The following examples can be prepared using similar sequences andmethods to those employed for the synthesis of Examples 16.

Intermediate 117a: (2R)-1-((tert-butyldimethylsilyl)oxy)pentadecan-3-yl1-methylpyrrolidine-2-carboxylate

Intermediate 1h (1.0 g, 2.79 mmol) was dissolved in DMF (30 mL).N-methyl-D-proline (0.54 g, 4.18 mmol), HATU (2.1 g, 5.58 mmol), andDIPEA (2.9 mL, 16.73 mL) were added, followed by DMAP (0.34 g, 2.79mmol) and the mixture was stirred for 18 h at room temperature. Themixture was diluted with water (200 mL), and extracted with EtOAc (2×100mL). The organic layers were combined, washed with brine (100 mL), driedover sodium sulfate, and concentrated to obtain a pale green oil. Thecrude mixture was purified by flash column chromatography over neutralalumina, eluting with 8% EtOAc/n-hexanes, to give 1.0 g of the desiredproduct.

TLC (silica gel, 10% MeOH/DCM, PMA stain): R_(f)=0.46.

Intermediate 117b: (2R)-1-hydroxypentadecan-3-yl1-methylpyrrolidine-2-carboxylate

Intermediate 117a (0.60 g, 1.27 mmol) was dissolved in THF (50 mL) andthe mixture was cooled to 0° C. HF.pyridne (4.5 mL of a 70% solution)was added dropwise, and the mixture was stirred for 1 h at 0° C. Themixture was then diluted with water (30 mL), and neutralized with solidNaHCO₃. This mixture was extracted with DCM (2×50 mL), and the organiclayers were combined, washed with brine (100 mL), dried over sodiumsulfate, and concentrated. The crude mixture was used in the next stepwithout further purification.

TLC (silica gel, 10% MeOH/DCM, PMA stain): R_(f)=0.28.

The following examples can be prepared using similar methods to thoseemployed for the synthesis of Examples 16, employing alcohols similar tointermediate 117b.

Example 117 (2R)-1-((6,6-bis(octyloxy)hexanoyl)oxy) pentadecan-3-yl1-methyl pyrrolidine-2-carboxylate

¹H NMR (400 MHz, CD₂Cl₂) δ: 5.04-4.94 (m, 1H), 4.41 (t, J=5.7 Hz, 1H),4.13-3.99 (m, 2H), 3.53 (dt, J=9.3, 6.7 Hz, 2H), 3.37 (dt, J=9.3, 6.7Hz, 2H), 3.11-3.00 (m, 1H), 2.96-2.85 (m, 1H), 2.35 (s, 3H), 2.32-2.23(m, 3H), 2.17-2.01 (m, 1H), 1.96-1.68 (m, 5H), 1.66-1.47 (m, 10H),1.40-1.20 (m, 42H), 0.93-0.83 (m, 9H).

MS (M+1)=710.7, Rt=2.70 min (LC method 13).

Example 118 (2S)-1-((6,6-bis(octyloxy) hexanoyl)oxy)pentadecan-3-yl1-methylpyrrolidine-2-carboxylate

¹H NMR (400 MHz, CD₂Cl₂) δ: 5.04-4.95 (m, 1H), 4.41 (t, J=5.6 Hz, 1H),4.12-3.99 (m, 2H), 3.53 (dt, J=9.3, 6.7 Hz, 2H), 3.37 (dt, J=9.3, 6.7Hz, 2H), 3.12-3.02 (m, 1H), 2.93 (br s, 1H), 2.36 (s, 3H), 2.33-2.23 (m,3H), 2.18-2.02 (m, 1H), 1.96-1.81 (m, 4H), 1.82-1.71 (m, 1H), 1.66-1.47(m, 11H), 1.40-1.20 (m, 41H), 0.93-0.83 (m, 9H). MS (M+1)=711.1, Rt=2.77min (LC method 13).

Example 119 (2R)-1-((6,6-bis(octyloxy) hexanoyl)oxy)pentadecan-3-ylpyrrolidine-2-carboxylate

¹H NMR (400 MHz, CD₂Cl₂) δ: 5.03-4.91 (m, 1H), 4.41 (t, J=5.6 Hz, 1H),4.13-4.00 (m, 2H), 3.77-3.69 (m, 1H), 3.53 (dt, J=9.3, 6.7 Hz, 2H), 3.37(dt, J=9.3, 6.7 Hz, 2H), 3.10-3.00 (m, 1H), 2.94-2.83 (m, 1H), 2.76-2.34(br s, 1H), 2.28 (t, J=7.5 Hz, 2H), 2.17-2.05 (m, 1H), 1.97-1.66 (m,5H), 1.66-1.47 (m, 10H), 1.40-1.18 (m, 42H), 0.94-0.82 (m, 9H).

MS (M+2)=697.3, Rt=2.58 min (LC method 13).

Example 120 1-((6,6-bis(octyloxy)hexanoyl)oxy) pentadecan-3-yl 1,3-dimethylpyrrolidine-3-carboxylate

¹H NMR (400 MHz, CDCl₃): δ=4.98 (m, 1H), 4.42 (t, J=8 Hz, 1H), 4.10 (q,J=8 Hz, 2H), 3.48 (q, J=8 Hz, 2H), 3.40 (q, J=4 Hz, 2H), 2.88 (d, J=8Hz, 1H), 2.68-2.62 (m, 1H), 2.58-2.45 (m, 3H), 2.34-2.24 (m, 4H),1.96-1.82 (m, 2H), 1.52 (m, 7H), 1.42-1.20 (m, 50H), 0.98-0.82 (m, 9H)ppm.

MS (M+1)=725.3, Rt=2.78 min (LC method 13).

Example 121 3-((3-(1-methylpiperidin-4-yl)propanoyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate

¹H NMR (400 MHz, CDCl₃): δ=4.98 (m, 1H), 4.42 (m, 1H), 4.10 (m, 2H),3.58-3.50 (m, 2H), 3.42-3.38 (m, 2H), 2.84 (m, 2H), 2.38-2.22 (m, 8H),1.98-1.82 (m, 5H), 1.76-142 (m, 10H), 1.38-1.20 (m, 47H), 0.84-0.80 (m,9H) ppm.

MS (M+1)=753.4, Rt=2.83 min (LC method 13).

Example 122 1-((6,6-bis(octyloxy)hexanoyl)oxy) pentadecan-3-yl1,4-dimethylpiperidine-4-carboxylate

¹H NMR (400 MHz, CD₂Cl₂) δ=4.93-5.02 (m, 1H), 4.41 (t, J=5.62 Hz, 1H),4.01-4.11 (m, 2H), 3.53 (dt, J=9.29, 6.66 Hz, 2H), 3.37 (dt, J=9.29,6.66 Hz, 2H), 2.51-2.61 (m, 2H), 2.29 (t, J=7.52 Hz, 2H), 2.20 (s, 3H),2.08 (dd, J=10.70, 3.48 Hz, 4H), 1.80-1.93 (m, 2H), 1.42-1.67 (m, 12H),1.20-1.39 (m, 42H), 1.17 (s, 3H), 0.83-0.93 (m, 9H) ppm.

MS (M+1)=739.2, Rt=2.81 min (LC method 13).

Example 123 3-((5-(diethylamino)pentanoyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate

¹H NMR (400 MHz, CD₂Cl₂) δ=4.98 (s, 1H), 4.45 (t, J=5.69 Hz, 1H),4.03-4.14 (m, 2H), 3.57 (dt, J=9.29, 6.66 Hz, 2H), 3.41 (dt, J=9.29,6.66 Hz, 2H), 2.40-2.59 (m, 6H), 2.32 (q, J=7.30 Hz, 4H), 1.81-1.95 (m,2H), 1.45-1.70 (m, 14H), 1.23-1.45 (m, 42H), 1.03 (t, J=7.03 Hz, 6H),0.87-0.97 (m, 9H) ppm.

MS (M+1)=755.2, Rt=2.68 min (LC method 13).

Synthesis of Example 124 Intermediate 124a:1-((tert-butyldimethylsilyl)oxy)decan-3-ol

Intermediate 1g (22.5 g, 119.6 mmol) was dissolved in THF (225 mL) andcooled to 0° C. Heptyl magnesium bromide (1M in diethyl ether, 143.5 mL,143.5 mmol) was added dropwise, and the mixture was stirred for 2 h at0° C. The reaction was quenched with the addition of aqueous ammoniumchloride (100 mL) and extracted with EtOAc (2×200 mL). The organiclayers were dried over sodium sulfate and concentrated to obtain thecrude product. The crude mixture was purified by flash columnchromatography over silica gel, eluting with 3% EtOAc/heptane to provide12.8 g of the desired product.

TLC (silica gel, 20% EtOAc/heptane, PMA stain): R_(f)=0.55.

Intermediate 124b:5-heptyl-2,2,9,9,10,10-hexamethyl-3,3-diphenyl-4,8-dioxa-3,9-disilaundecane

Intermediate 124a (12.8 g, 44.4 mmol) was dissolved in DCM (130 mL).Imidazole (4.53 g, 66.6 mmol) and DMAP (0.54 g, 4.44 mmol) were added,followed by TBDPSCl (11.55 mL, 44.4 mmol), and the mixture was stirredfor 16 h at room temperature. The mixture was then diluted with water(200 mL) and extracted with DCM (2×100 mL). The organic layers werecombined, washed with brine (100 mL), dried over sodium sulfate, andconcentrated. The crude mixture was purified by flash columnchromatography over silica gel, eluting with 1% EtOAc/heptane to provide14.5 g of the desired product.

TLC (silica gel, 10% EtOAc/heptane, UV): R_(f)=0.96.

Intermediate 124c: 3-((tert-butyldiphenylsilyl)oxy)decan-1-ol

Intermediate 124b (14.5 g, 27.5 mmol) was dissolved in ethanol (145 mL)and treated with PPTS (7.61 g, 30.3 mmol). The mixture was stirred for16 h at room temperature. The mixture was then diluted with water (300mL) and extracted with DCM (2×100 mL). The organic layers were combined,washed with brine (100 mL), dried over sodium sulfate, and concentrated.The crude material was used in the next step without furtherpurification TLC (silica gel, 10% EtOAc/heptane, UV): R_(f)=0.31.

Intermediate 124d: 3-((tert-butyldiphenylsilyl)oxy)decanal

IBX (14.4 g, 51.5 mmol) was dissolved in DMSO (70 mL) and intermediate124c (8.5 g, 20.6 mmol) was added. The mixture was stirred for 2 h atroom temperature. The reaction mixture was diluted with diethyl ether(500 mL) and the resulting solid was filtered, and the filtrate waswashed with brine (2×100 mL), dried over sodium sulfate, andconcentrated. The crude product was used in the next step withoutfurther purification.

TLC (silica gel, 10% EtOAc/heptane, UV): R_(f)=0.66.

Intermediate 124e: 10-((tert-butyldiphenylsilyl)oxy)heptadecan-8-ol

Intermediate 124d (8.0 g, 19.5 mmol) was dissolved in THF (80 mL) andcooled to 0° C. Heptyl magnesium bromide (1M in diethyl ether, 23.4 mL,23.4 mmol) was added dropwise, and the mixture was stirred for 2 h at 0°C. The reaction was quenched with the addition of aqueous ammoniumchloride (100 mL) and extracted with EtOAc (2×100 mL). The organiclayers were dried over sodium sulfate and concentrated to obtain thecrude product. The crude mixture was purified by flash columnchromatography over silica gel, eluting with 1% EtOAc/heptane to provide6.0 g of the desired product.

TLC (silica gel, 10% EtOAc/heptane, UV): R_(f)=0.52.

Intermediate 124f: heptadecane-8,10-diol

Intermediate 124e (6.0 g, 11.7 mmol) was dissolved in THF (60 mL) andcooled to 0° C. HF pyridine (70%, 3.39 mL) was added dropwise, then themixture was warmed to room temperature and stirred for 16 h. The mixturewas diluted with water (100 mL) and extracted with DCM (2×50 mL). Thecombined organic layers were washed with brine (100 mL) dried oversodium sulfate and concentrated to obtain the crude product. The crudemixture was then triturated with pentane to provide 1.1 g of the desiredproduct.

TLC (silica gel, 10% EtOAc/heptane, PMA stain): R_(f)=0.51.

Intermediate 124g: methyl 5-(4,6-diheptyl-1,3-dioxan-2-yl)pentanoate

Intermediate 16a (1.0 g, 5.2 mmol), intermediate 124f (2.1 g, 7.9 mmol)and KHSO₄ were combined neat, and heated to 70° C. for 4 h. The mixturewas cooled to room temperature, diluted with water (40 mL), andextracted with EtOAc (2×50 mL). The combined organic layers were washedwith brine (100 mL), dried over sodium sulfate, and concentrated. Thecrude mixture was purified by flash column chromatography over silicagel, eluting with 4% EtOAc/heptane to provide 1.1 g of the desiredproduct.

TLC (silica gel, 10% EtOAc/heptane, PMA stain): R_(f)=0.47.

Intermediate 124h: 5-(4,6-diheptyl-1,3-dioxan-2-yl)pentanoic acid

Intermediate 124g (1.0 g, 2.5 mmol) was dissolved in water/methanol(1:1, 40 mL). NaOH (0.5 g, 12.5 mmol) was added and the mixture washeated to reflux for 2 h. The mixture was then cooled to roomtemperature, and neutralized with 1N HCl. The mixture was then extractedwith EtOAc (2×50 mL), and the organic layers were combined, washed withbrine (100 mL), dried over sodium sulfate, and concentrated. The crudematerial was used in the next step without further purification.

TLC (silica gel, 10% EtOAc/heptane, PMA stain): R_(f)=0.14.

The following example can be prepared using similar sequences andmethods to those employed for the synthesis of Example 16.

Example 124 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl5-(4,6-diheptyl-1,3-dioxan-2-yl)pentanoate

¹H NMR (400 MHz, CD₂Cl₂) δ: 4.81-4.74 (m, 1H), 4.73 (t, J=5.1 Hz, 1H),4.20-4.03 (m, 4H), 4.00-3.91 (m, 1H), 3.76-3.66 (m, 1H), 2.53-2.41 (m,6H), 2.28 (t, J=7.6 Hz, 2H), 1.99-1.84 (m, 3H), 1.81-1.67 (m, 3H),1.64-1.54 (m, 4H), 1.54-1.42 (m, 4H), 1.42-1.20 (m, 44H), 0.98 (t, J=7.1Hz, 6H), 0.92-0.84 (m, 9H).

MS (M+1)=769.2, Rt=2.73 min (LC method 13).

Synthesis of Example 125 Intermediate 125a:1-((tert-butyldimethylsilyl)oxy)undecan-3-ol

To a solution of intermediate 1g 4.0 g, 21.2 mmol) in Et₂O (40 mL) in aRBF charged with a magnetic stir bar under N₂ was added octyl magnesiumbromide (2 M in Et₂O, 12.6 mL, 25.5 mmol) dropwise at 0° C. Theresulting mixture was stirred for 2 hours at 0° C., after which thereaction was quenched with aqueous saturated NH₄Cl (100 mL) andextracted with EtOAc (2×200 mL). The combined organic layers were driedover Na₂SO₄ and concentrated under reduced pressure to obtain a crudeliquid. The crude product was purified by silica gel chromatographyeluting with EtOAc:hexane (product eluted at 3% EtOAc:hexane) to affordthe desired product as a pale yellow liquid (1.8 g).

TLC: Rf=0.7 (EtOAc: Hexane, 2:8), PMA active.

Intermediate 125b: 1-((tert-butyldimethylsilyl)oxy)undecan-3-yl(3-(diethylamino)propyl) carbonate

To a stirred solution of intermediate 125a (1.8 g, 5.9 mmol) in DCM (18mL) in a RBF charged with a magnetic stir bar under N₂ was addedpyridine (2.4 mL, 29.7 mmol) and DMAP (363 mg, 2.9 mmol), followed by4-nitrophenyl chloroformate (2.3 g, 11.9 mmol) at rt. The reaction wasstirred for 8 hours, after which 3-(diethylamino)propan-1-ol (1.5 g,11.9 mmol) was added. The reaction was stirred at rt for 48 hours, afterwhich it was diluted with H₂O and extracted with DCM (2×100 mL). Thecombined organic layers were dried over Na₂SO₄ and concentrated underreduced pressure to obtain a yellow liquid. The crude product waspurified by silica gel chromatography eluting with EtOAc:hexane (producteluted at 2% EtOAc:hexane) to afford the desired product as a yellowliquid (1.45 g).

TLC: Rf=0.6 (EtOAc: Hexane, 2:8), PMA active.

Intermediate 125c: 3-(diethylamino)propyl (1-hydroxyundecan-3-yl)carbonate

To a 0° C. solution of intermediate 125b (1.0 g, 2.1 mmol) in THF (20mL) in a round bottom flask charged with a magnetic stir bar under N₂was added HF-pyridine (30-70%, 3.1 mL). The resulting mixture wasstirred for 1 hour at 0° C., after which it was diluted with H₂O (30mL), neutralized with solid NaHCO₃, and extracted with DCM (2×50 mL).The combined organic layers were washed with brine (100 mL), dried overNa₂SO₄, and concentrated under reduced pressure to obtain the desiredproduct as a crude pale green liquid (730 mg). The crude material wasused for the next step without purification.

TLC: Rf=0.4 (MeOH:DCM, 1:9), PMA active.

Example 125 3-(((3-(diethylamino)propoxy)carbonyl)oxy)undecyl6,6-bis(octyloxy)hexanoate

To a solution of intermediate 125c (730 mg, 2.1 mmol) in DMF (30 mL) ina RBF charged with a magnetic stir bar under N₂ were added intermediate16c (1.10 g, 3.11 mmol), EDC.HCl (1.20 g, 6.35 mmol), DIPEA (2.2 mL,12.7 mmol), and DMAP (258 mg, 2.11 mmol). The resulting mixture wasstirred at 25-30° C. for 18 hours, after which it was diluted with H₂O(200 mL) and extracted with EtOAc (2×100 mL). The combined organiclayers were washed with brine (100 mL), dried over Na₂SO₄, andconcentrated under reduced pressure to obtain a crude pale green liquid.The crude product was purified by flash chromatography over neutralalumina, eluting with EtOAc:hexane (product eluted at 8% EtOAc:hexane)to afford the desired product as a colorless liquid (250 mg).

¹H NMR (400 MHz, CD₂Cl₂) δ=4.77 (quin, J=6.25 Hz, 1H), 4.41 (t, J=5.62Hz, 1H) 4.02-4.20 (m, 4H) 3.53 (dt, J=9.22, 6.69 Hz, 2H), 3.37 (dt,J=9.22, 6.69 Hz, 2H), 2.49 (d, J=6.06 Hz, 6H), 2.28 (t, J=7.52 Hz, 2H),1.89 (q, J=6.40 Hz, 2H), 1.71-1.82 (m, 2H), 1.46-1.67 (m, 10H),1.20-1.42 (m, 34H), 0.99 (t, J=7.01 Hz, 6H), 0.88 (t, J=6.63 Hz, 9H)ppm.

MS (M+1)=701.1, Rt=2.33 min (LC method 13).

Synthesis of Example 126 Intermediate 126a:1-((tert-butyldimethylsilyl)oxy)tridecan-3-ol

To a solution decyl magnesium bromide (1M in Et₂O, 26 mL) in a RBFcharged with a magnetic stir bar under N₂ at 0° C. was addedintermediate 1g (4.0 g, 21.2 mmol) in Et₂O (70 mL). The resultingmixture was stirred for 2 hours at 0° C., after which it was slowlyquenched with saturated NH₄Cl solution (100 mL) at 0° C. and extractedwith EtOAc (2×250 mL). The combined organic layers were again washedwith saturated NH₄Cl solution (250 mL), dried over Na₂SO₄, andevaporated to dryness to obtain a colorless crude liquid. The crudeproduct was purified by silica gel chromatography eluting withEtOAc:hexane (product eluted at 5% EtOAc:hexane) to afford the desiredproduct as a colorless liquid (4.0 g).

TLC: Rf=0.6 (EtOAc: Hexane, 1:9), PMA active.

Intermediate 126b: 1-((tert-butyldimethylsilyl)oxy)tridecan-3-yl2-(4-nitrophenyl)acetate

To a solution of intermediate 126a (1.5 g, 4.54 mmol) in DCM (30 mL) ina RBF charged with a magnetic stir bar under N₂ was added pyridine (1.2mL, 13.6 mmol) followed by 4-nitrophenyl chloroformate (1.37 g, 6.81mmol) and DMAP (1.7 g, 13.6 mmol) at 0° C. The reaction was stirred for2 hours at 30° C., after which it was quenched with H₂O (50 mL) andextracted with EtOAc (3×100 mL). The combined organic layers were driedover Na₂SO₄ and evaporated to dryness to obtain a crude pale yellowliquid. The crude product was purified by silica gel chromatographyeluting with EtOAc:hexane (product eluted at 2% EtOAc:hexane) to affordthe desired product as a pale yellow liquid (1.8 g).

TLC: Rf=0.9 (EtOAc: Hexane, 0.5:9.5), PMA active.

Intermediate 126c: 1-((tert-butyldimethylsilyl)oxy)tridecan-3-yl(3-(diethylamino)propyl) carbonate

To a solution of intermediate 126b (1.8 g, 3.63 mmol) in DCM (15 mL) ina RBF charged with a magnetic stir bar under N₂ was added pyridine (0.6mL, 7.26 mmol) followed by 3-(diethylamino)propan-1-ol (1.0 mL, 7.26mmol) and DMAP (900 mg, 7.26 mmol). The reaction was stirred at 30° C.for 16 hours, after which it was quenched with H₂O (50 mL) and extractedwith DCM (3×100 mL). The combined organic layers were dried over Na₂SO₄and evaporated to dryness to obtain a crude pale yellow liquid. Thecrude product was purified by silica gel chromatography eluting withMeOH:DCM (product eluted at 4% MeOH:DCM) to afford the desired productas a pale yellow liquid (1.4 g).

TLC: Rf=0.4 (MeOH:DCM, 1:9), PMA active.

Intermediate 126d: 3-(diethylamino)propyl (1-hydroxytridecan-3-yl)carbonate

To a solution of intermediate 126c (1.4 g, 2.87 mmol) in THF (10 mL) ina round bottom flask charged with a magnetic stir bar under N₂ at 0° C.was added HF-pyridine (4.9 mL, 60 equiv.). The resulting mixture wasstirred for 1 hour, after which it was slowly basified with saturatedNaHCO₃ and extracted with EtOAc (2×50 mL). The combined organic layerswere dried over Na₂SO₄ and evaporated to dryness to obtain the desiredproduct as a crude pale yellow oil (1.0 g). This compound was used inthe next step without further purification.

TLC: Rf=0.4 (MeOH:DCM, 1:9), PMA active.

Example 126 3-(((3-(diethylamino)propoxy)carbonyl)oxy)tridecyl6,6-bis(octyloxy)hexanoate

To a solution of intermediate 126d (1.00 g, 2.67 mmol) and intermediate16c (1.20 g, 3.21 mmol) in DMF (10 mL) in a RBF charged with a magneticstir bar under N₂ were added HATU (2.0 g, 5.35 mmol), DIPEA (1.4 mL,8.034 mmol), and DMAP (165 mg, 1.33 mmol). The reaction was stirred at30° C. under N₂ for 16 hours, after which it was quenched with H₂O (100mL) and extracted with EtOAc (3×200 mL). The combined organic layerswere washed with brine, dried over Na₂SO₄, and evaporated to dryness toobtain a crude pale yellow liquid. The crude product was purified byneutral alumina chromatography eluting with EtOAc:hexane (product elutedat 6% EtOAc:hexane) to afford the desired product as a pale yellowliquid (500 mg).

¹H NMR (400 MHz, CD₂Cl₂) δ=4.77 (quin, J=6.22 Hz, 1H), 4.41 (t, J=5.62Hz, 1H), 4.02-4.21 (m, 4H), 3.53 (dt, J=9.22, 6.69 Hz, 2H), 3.37 (dt,J=9.22, 6.69 Hz, 2H), 2.49 (br. s., 6H), 2.28 (t, J=7.52 Hz, 2H), 1.90(q, J=6.40 Hz, 2H), 1.78 (br. s., 2H), 1.46-1.67 (m, 10H), 1.20-1.41 (m,38H), 1.00 (t, J=6.69 Hz, 6H), 0.88 (t, J=6.57 Hz, 9H) ppm.

MS (M+1)=729.2, Rt=2.60 min (LC method 13).

Synthesis of Example 127 Intermediate 127a:(6Z,9Z)-18-bromooctadeca-6,9-diene

To a suspension of magnesium bromide diethyl etherate (22.5 g, 87.1mmol) in Et₂O (250 mL) in a RBF charged with a magnetic stir bar underN₂ was added linoleoyl mesylate (15 g, 43.5) slowly. The reaction wasstirred vigorously for 40 min, after which it was quenched with ice coldH₂O (200 mL). The organic layer was separated and the aqueous layer wasextracted with Et₂O (2×250 mL). The combined organic layers were driedover Na₂SO₄ and evaporated to dryness to afford the desired product as acrude pale yellow oil (14.0 g, 98%). This material was used in the nextstep without further purification.

TLC: Rf=0.9 (EtOAc:hexane, 2:8), PMA active.

Intermediate 127b: (6Z,9Z)-18-iodooctadeca-6,9-diene

To a solution of intermediate 127a (14.0 g, 42.659 mmol) in acetone (150mL) in a RBF charged with a magnetic stir bar under N₂ was added sodiumiodide (12.7 g, 85.3 mmol). The reaction was heated to reflux (55° C.)for 2 hours, after which it was cooled to room temperature and thesolids were filtered off. The solvent was evaporated and the remainingsolids were removed by dissolution in H₂O (100 mL) and extraction withDCM (2×200 mL). The combined organic layers were dried over Na₂SO₄ andevaporated to dryness to afford the desired product as a crude palebrown liquid (15.5 g, 97%). This material was used in the next stepwithout further purification.

TLC: Rf=0.6 (100% pentane), PMA active.

Intermediate 127c:(12Z,15Z)-1-((tert-butyldimethylsilyl)oxy)henicosa-12,15-dien-3-ol

To a solution of intermediate 127b (15.0 g, 39.8 mmol) in Et₂O (80 mL)and pentane (20 mL) in a RBF charged with a magnetic stir bar under N₂was added t-Butyl lithium (1.5 M, 53 mL, 79.7 mmol) at −78° C. Next,intermediate 1g (7.5 g, 39.8 mmol) was added as a solution in 20 mLEt₂O. The reaction was stirred at −78° C. for 10 minutes, after which itwas quenched with saturated NH₄Cl solution (100 mL). The organic layerwas separated and the aqueous layer was extracted with Et₂O (150 mL).The combined organic layers were dried over Na₂SO₄ and evaporated todryness to obtain a crude colorless viscous liquid. The crude productwas purified by silica gel chromatography eluting with EtOAc:hexane(product eluted at 4% EtOAc:hexane) to afford the desired product as acolorless viscous liquid (6.0 g).

TLC: Rf=0.5 (EtOAc: Hexane, 1:9), PMA active.

Intermediate 127d:(12Z,15Z)-1-((tert-butyldimethylsilyl)oxy)henicosa-12,15-dien-3-yl(4-nitrophenyl) carbonate

To a solution of intermediate 127c (1.5 g, 3.41 mmol) in DCM (30 mL) ina RBF charged with a magnetic stir bar under N₂ was added pyridine (0.9mL, 10.251 mmol) followed by 4-nitrophenyl chloroformate (1.0 g, 5.12mmol) and DMAP (1.2 g, 10.2 mmol) at 0° C. The reaction was stirred for2 hours at 30° C., after which it was quenched with H₂O (50 mL) andextracted with EtOAc (3×100 mL). The combined organic layers were driedover Na₂SO₄ and evaporated to dryness to obtain a crude pale yellowliquid. The crude product was purified by silica gel chromatographyeluting with EtOAc:hexane (product eluted at 2% EtOAc:hexane) to affordthe desired product as a pale yellow liquid (1.6 g).

TLC: Rf=0.9 (EtOAc: Hexane, 0.5:9.5), PMA active.

Intermediate 127e:(12Z,15Z)-1-((tert-butyldimethylsilyl)oxy)henicosa-12,15-dien-3-yl(3-(diethylamino)propyl) carbonate

To a solution of intermediate 127d (1.6 g, 2.65 mmol) in DCM (15 mL) ina RBF charged with a magnetic stir bar under N₂ was added pyridine (0.5mL, 5.30 mmol) followed by 3-(diethylamino)propan-1-ol (0.8 mL, 5.30mmol) and DMAP (650 mg, 5.30 mmol). The reaction was stirred at 30° C.for 16 hours, after which it was quenched with H₂O (50 mL) and extractedwith DCM (3×100 mL). The combined organic layers were dried over Na₂SO₄and evaporated to dryness to obtain a crude pale yellow liquid. Thecrude product was purified by silica gel chromatography eluting withMeOH:DCM (product eluted at 4% MeOH:DCM) to afford the desired productas a pale yellow liquid (1.1 g).

TLC: Rf=0.97 (MeOH:DCM, 1:9), PMA active.

Intermediate 127f: 3-(diethylamino)propyl((12Z,15Z)-1-hydroxyhenicosa-12,15-dien-3-yl) carbonate

To a solution of intermediate 127e (1.1 g, 2.01 mmol) in THF (10 mL) ina round bottom flask charged with a magnetic stir bar under N₂ at 0° C.was added HF-pyridine (3.5 mL, 60 equiv.). The resulting mixture wasstirred at 0° C. for 1 hour, after which it was slowly basified withsaturated NaHCO₃ and extracted with EtOAc (2×50 mL). The combinedorganic layers were dried over Na₂SO₄ and evaporated to dryness toafford the desired product as a crude pale yellow oil (900 mg). Thiscompound was used in the next step without further purification.

TLC: Rf=0.4 (MeOH:DCM, 1:9), PMA active.

Example 127 (12Z,15Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)henicosa-12,15-dien-1-yl 6,6-bis(octyloxy)hexanoate

To a solution of intermediate 127f (900 mg, 1.86 mmol) and intermediate16c (1.4 g, 3.738 mmol) in DMF (10 mL) in a RBF charged with a magneticstir bar under N₂ were added HATU (1.4 g, 3.73 mmol), DIPEA (1.0 mL,5.607 mmol), and DMAP (115 mg, 0.93 mmol). The reaction was stirred at30° C. under N₂ for 16 hours, after which it was quenched with H₂O (100mL) and extracted with EtOAc (3×200 mL). The combined organic layerswere washed with brine, dried over Na₂SO₄, and evaporated to dryness toafford a crude pale yellow liquid. The crude product was purified byneutral alumina chromatography eluting with EtOAc:hexane (product elutedat 7% EtOAc:hexane) to afford the desired product as a pale yellowliquid (550 mg).

¹H NMR (400 MHz, CD₂Cl₂) δ=5.25-5.45 (m, 4H), 4.77 (quin, J=6.24 Hz,1H), 4.41 (t, J=5.62 Hz, 1H), 4.04-4.19 (m, 4H), 3.53 (dt, J=9.29, 6.66Hz, 2H), 3.37 (dt, J=9.29, 6.66 Hz, 2H), 2.78 (t, J=6.54 Hz, 2H), 2.50(br. s., 6H), 2.29 (t, J=7.58 Hz, 2H), 2.05 (q, J=6.81 Hz, 4H), 1.90 (q,J=6.44 Hz, 2H), 1.77 (br. s., 2H), 1.45-1.67 (m, 10H), 1.21-1.41 (m,40H), 1.01 (br. s., 6H), 0.83-0.95 (m, 9H) ppm.

MS (M+1)=837.3, Rt=1.20 min (LC method 14).

Synthesis of Example 128 Intermediate 128a:6-(benzyloxy)-1-((tert-butyldimethylsilyl)oxy)hexan-3-ol

To a solution of magnesium (1.76 g, 73.6 mmol) and I₂ (100 mg,catalytic) in THF (50 mL) in a RBF (fitted with a reflux condenser)charged with a magnetic stir bar under N₂ was added benzyl 3-bromopropylether (14.0 g, 61.4 mmol) in THF (40 mL). The reaction was stirred for 1hour, after which it was added to intermediate 1g (13.85 g, 73.6 mmol)in THF (60 mL) at 0° C. The reaction was allowed to warm to 25° C. for 1hour and stir at that temperature for 17 hours, after which it wasquenched with saturated NH₄Cl (150 mL) and extracted with EtOAc (2×250mL). The combined organic layers were dried over Na₂SO₄ and concentratedunder reduced pressure to obtain a crude brown liquid. The crude productwas purified by silica gel chromatography eluting with EtOAc:hexane(product eluted at 15% EtOAc:hexane) to afford the desired product as apale yellow liquid (2.3 g).

TLC: Rf=0.2 (EtOAc: Hexane, 2:8), PMA active.

Intermediate 128b:6-(benzyloxy)-1-((tert-butyldimethylsilyl)oxy)hexan-3-yl (4-nitrophenyl)carbonate

To a solution of intermediate 128a (2.0 g, 5.91 mmol) in DCM (40 mL) ina RBF charged with a magnetic stir bar under N₂ was added 4-nitrophenylchloroformate (1.78 g, 8.86 mmol) followed by pyridine (1.45 mL, 17.7mmol) at 0° C. The reaction was stirred for 18 hours, after which it wasdiluted with H₂O (100 mL) and extracted with DCM (2×100 mL). Thecombined organic layers were washed with brine (100 mL), dried overNa₂SO₄, and concentrated under reduced pressure to obtain a crude gummysolid. The crude product was purified by silica gel chromatographyeluting with EtOAc:hexane (product eluted at 5% EtOAc:hexane) to affordthe desired product as a pale brown liquid (400 mg).

TLC: Rf=0.6 (EtOAc: Hexane, 1:9), UV & PMA active.

Intermediate 128c:6-(benzyloxy)-1-((tert-butyldimethylsilyl)oxy)hexan-3-yl(3-(diethylamino)propyl) carbonate

To a solution of intermediate 128b (1.9 g, 3.77 mmol) in DCM (40 mL) ina RBF charged with a magnetic stir bar under N₂ were added3-(diethylamino)propan-1-ol (2.81 mL, 18.8 mmol) followed by Et₃N (5.27mL, 37.7 mmol) and DMAP (922 mg, 7.55 mmol). The reaction was stirred atrt for 3 days, after which it was diluted with H₂O (200 mL) andextracted with DCM (2×200 mL). The combined organic layers were washedwith brine (250 mL), dried over Na₂SO₄, and concentrated under reducedpressure to obtain a crude pale green liquid. The crude product waspurified by silica gel chromatography eluting with MeOH:DCM (producteluted at 3% MeOH:DCM) to afford the desired product as a pale greenliquid (550 mg).

TLC: Rf=0.6 (MeOH:DCM, 1:9), PMA active.

Intermediate 128d: 6-(benzyloxy)-1-hydroxyhexan-3-yl(3-(diethylamino)propyl) carbonate

To a solution of intermediate 128c (550 mg, 1.11 mmol) in THF (20 mL) ina round bottom flask charged with a magnetic stir bar under N₂ at 0° C.was added HF-pyridine (30-70%, 2.0 mL). The resulting mixture wasstirred at 0° C. for 1 hour, after which it was diluted with H₂O (30mL), neutralized with solid NaHCO₃, and extracted with DCM (2×50 mL).The combined organic layers were washed with brine (100 mL), dried overNa₂SO₄, and concentrated under reduced pressure to afford the desiredproduct as a crude pale green liquid (400 mg).

TLC: Rf=0.4 (MeOH:DCM, 1:9), I₂ & PMA active.

Intermediate 128e:6-(benzyloxy)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)hexyl6,6-bis(octyloxy)hexanoate

To a solution of intermediate 128d (400 mg, 1.04 mmol) in DMF (15 mL) ina RBF charged with a magnetic stir bar under N₂ were added intermediate16c (585 mg, 1.57 mmol), HATU (797 mg, 2.09 mmol), DIPEA (0.55 mL, 3.147mmol), and DMAP (128 mg, 1.04 mmol) sequentially. The reaction wasstirred at rt for 18 hours, after which it was diluted with H₂O (100 mL)and extracted with EtOAc (2×50 mL). The combined organic layers werewashed with brine (100 mL), dried over Na₂SO₄, and concentrated underreduced pressure to afford a crude pale green liquid. The crude productwas purified by neutral alumina chromatography eluting with EtOAc:hexane(product eluted at 15% EtOAc:hexane) to afford the desired product as apale green liquid (550 mg).

TLC: Rf=0.6 (MeOH:DCM, 1:9), PMA active.

Intermediate 128f:3-(((3-(diethylamino)propoxy)carbonyl)oxy)-6-hydroxyhexyl6,6-bis(octyloxy)hexanoate

Raney Ni (20 mg, cat.) was added to a solution of intermediate 128e (60mg, 0.081 mmol) in EtOH (5 mL) in a RBF charged with a magnetic stirbar. The resulting mixture was stirred under atmospheric H₂ pressure(using a hydrogen balloon) for 24 hours at rt, after which it wasfiltered on a bed of celite and concentrated under reduced pressure toobtain the desired product as a colorless liquid (40 mg, 76%). Thiscompound was used in the next step without further purification.

TLC: Rf=0.4 (MeOH:DCM, 1:9), PMA active.

Example 128 6-((6,6-bis(octyloxy)hexanoyl)oxy)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexyl octanoate

To a solution of intermediate 128f (440 mg, 0.681 mmol) in DMF (15 mL)in a RBF charged with a magnetic stir bar under N₂ were added octanoicacid (0.22 mL, 1.36 mmol), HATU (777 mg, 2.04 mmol), DIPEA (0.59 mL,3.40 mmol), and DMAP (83 mg, 0.68 mmol) sequentially. The reaction wasstirred at rt for 18 hours, after which it was diluted with H₂O (100 mL)and extracted with EtOAc (2×50 mL). The combined organic layers werewashed with brine (100 mL), dried over Na₂SO₄, and concentrated underreduced pressure to afford a crude pale green liquid. The crude productwas purified by neutral alumina chromatography eluting with EtOAc:hexane(product eluted at 15% EtOAc:hexane) to afford the desired product as acolorless liquid (400 mg).

¹H NMR (400 MHz, CDCl₃): δ=4.82 (m, 1H), 4.33 (m, 1H), 4.20-4.08 (m,5H), 3.48 (m, 2H), 3.40 (m, 2H), 2.52 (m, 5H), 2.30 (q, J=8 Hz, 4H),1.92 (m, 2H), 1.82 (q, J=8 Hz, 2H), 1.78-1.50 (m, 11H), 1.39-1.22 (m,35H), 1.02 (t, J=8 Hz, 6H), 0.92-0.82 (m, 9H) ppm.

MS (M+1)=773.1, Rt=2.41 min (LC method 13).

Synthesis of Example 129 Intermediate 129a:1-((tert-butyldimethylsilyl)oxy)heptadecan-5-ol

To a stirred solution of 5-((tert-butyldimethylsilyl)oxy)pentanal (6.00g, 31.2 mmol) under nitrogen atmosphere at 0° C. was added 1 M dodecylmagnesium bromide (37 mL, 37 mmol) in dry diethyl ether (40 mL). Thereaction was stirred for 2 h at the same temperature. Progress of thereaction was monitored by TLC. The reaction mixture was slowly quenchedwith 100 mL of saturated ammonium chloride solution at 0° C. andextracted with (2×250 mL) ethyl acetate. The combined organic layerswere again washed with 250 mL of saturated ammonium chloride, dried oversodium sulfate and evaporated to dryness to get colorless liquid incrude form. The crude product was purified by flash chromatographyeluting with 5% EtOAc in hexanes to afford 5 g of colorless oil.

TLC: Rf=0.5 (EtOAc: hexanes, 2:8); PMA active.

Intermediate 129b: 1-((tert-butyldimethylsilyl)oxy)heptadecan-5-yl(4-nitrophenyl) carbonate

To a solution of intermediate 129a (2.50 g, 6.47 mmol) in DCM (15 mL)was added pyridine (2 mL, 25.9 mmol) and DMAP (10 mg) followed by slowaddition of 4-nitrophenyl chloroformate (2.6 g, 12.9 mmol) at 0° C. andthe reaction was stirred at 30° C. for 3 h. The reaction mixture wasquenched with 50 mL of water and extracted with DCM (3×50 mL). Thecombined organic layers were dried over sodium sulfate and evaporated todryness to afford pale yellow color liquid in crude form. The crudemixture was purified by flash chromatography eluting with 5% EtOAc inhexanes to afford 3.0 g of the desired product as a yellow oil.

TLC: Rf=0.8 (EtOAc:hexanes, 2:8); PMA active.

Intermediate 129c: 1-((tert-butyldimethylsilyl)oxy)heptadecan-5-yl(3-(diethylamino)propyl) carbonate

To a solution of intermediate 129b (3.0 g, 5.44 mmol) in DCM (6 mL) wasadded pyridine (1.7 mL, 21.76 mmol) and DMAP (332 mg, 2.72 mmol)followed by slow addition of 3-(diethylamino)propan-1-ol (1.6 mL, 10.9mmol) and the reaction was stirred at 30° C. for 16 h. The reactionmixture was quenched with 50 mL of water and extracted with DCM (3×50mL). The combined organic layers were dried over sodium sulfate andevaporated to dryness to afford pale yellow color liquid in crude form.The crude mixture was purified by flash-chromatography eluting with 4%MeOH in DCM to afford 2.5 g of the desired product as a yellow oil.

TLC: Rf=0.6 (MeOH:DCM, 1:9); PMA active.

Intermediate 129d: 3-(diethylamino)propyl (1-hydroxyheptadecan-5-yl)carbonate

To a solution of intermediate 129c (500 mg, 0.92 mmol) in THF (5 mL) wasadded HF.Pyridine complex (70%, 1.6 mL, 55.2 mmol) at 0° C. and thereaction was stirred for 1 h. The reaction mixture was slowly basifiedwith saturated sodium bicarbonate and extracted with (2×50 mL) EtOAc.The combined organic layers were dried over sodium sulfate andevaporated to dryness to afford 400 mg of the desired product as a paleyellow oil.

TLC: Rf=0.4 (MeOH:DCM, 1:9); PMA active.

Intermediate 129e: 3-(diethylamino)propyl (1-oxoheptadecan-5-yl)carbonate

To a solution of intermediate 129d (400 mg, 0.931 mmol) and triethylamine (1.2 mL, 8.37 mmol) in DCM (4.6 mL) was added sulfur trioxidepyridine complex (666 mg, 4.18 mmol) dissolved in DMSO (5.5 mL) at 0° C.The reaction was allowed to stir for 15 h at 30° C. The reaction mixturewas slowly quenched with 50 mL of water and extracted with (3×50 mL) ofEtOAc. The combined organic layers were dried over sodium sulfate andevaporated to dryness to afford pale yellow color liquid, 400 mg, incrude form. Material was used in the next step without furtherpurification.

TLC: Rf=0.5 (MeOH:DCM, 1:9); PMA active.

Intermediate 129f:5-(((3-(diethylamino)propoxy)carbonyl)oxy)heptadecanoic acid

To a stirred solution of intermediate 129e (400 mg, 0.93 mmol) int-Butanol (5 mL) and aq. buffer (pH 4.5, NaHPO₄:NaH₂PO₄:H₃PO₄, 1:1:1, 5mL) was added 2-methyl 2-butene (1.0 mL, 9.3 mmol) followed by additionof sodium chlorite (170 mg, 1.87 mmol) dissolved in 1.6 mL of water. Thethick hazy solution was stirred at 30° C. for 20 h. The reaction mixturewas quenched with 50 mL of water and extracted with (3×50 mL) EtOAc. Thecombined organic layers were dried over sodium sulfate and evaporated todryness to afford pale yellow color liquid, 450 mg, in crude form.

TLC: Rf=0.4 (MeOH:DCM, 1:9); PMA active.

Intermediate 129g: ((4,4-dimethoxybutoxy)methyl)benzene

To the mixture of 4-(benzyloxy)butanal (16.4 g, 92.13 mmol) andtrimethyl orthoformate (50 mL) in methanol (100 mL) was added sulfuricacid (200 mg) and the reaction was heated to 80° C. for 16 h undernitrogen atmosphere. Solvent was evaporated under vacuum to give a thickcrude liquid which was purified by chromatography eluting with at 10%EtOAc in hexanes to afford 12 g of colorless oil.

TLC: Rf=0.6 (EtOAc: hexanes, 2:8); PMA active.

Intermediate 129h: ((4,4-bis(octyloxy)butoxy)methyl)benzene

To a mixture of intermediate 129g (12.0 g, 53.53 mmol) and 1-octanol(28.0 g, 214.15 mmol) in benzene (50 mL) was added KHSO₄ (1 g). Thereaction was heated to 70° C. for 4 h under a nitrogen atmosphere. Thereaction mixture was allowed to cool to room temperature and waspurified directly by flash column chromatography, eluting with 4% EtOAcin hexanes to give 7.5 g of the desired product.

TLC: Rf=0.9 (EtOAc:hexanes, 2:8); PMA active.

Intermediate 129i: 4,4-bis(octyloxy)butan-1-ol

To a solution of intermediate 129h (3.0 g, 7.13 mmol) in EtOH (50 mL)was added Raney Nickel (6.0 g) and the reaction was stirred underhydrogen balloon pressure for 7 days at 30° C. The Reaction mixture wasfiltered through a celite bed and solvent was evaporated to dryness togive 2 g of colorless oil.

TLC: Rf=0.5 (EtOAc: hexanes, 2:8); PMA active.

The following example can be prepared using similar sequences andmethods to those employed for the synthesis of Example 11.

Example 129 4,4-bis(octyloxy)butyl5-(((3-(diethylamino)propoxy)carbonyl)oxy)heptadecanoate

¹H NMR (400 MHz, CD₂Cl₂) δ=4.67 (quin, J=5.70 Hz, 1H), 4.45 (m, 1H),4.14 (td, J=6.48, 1.71 Hz, 2H), 3.99-4.07 (m, 2H), 3.54 (m, 2H), 3.38(m, 2H), 2.55 (m, 4H), 2.26-2.35 (m, 2H), 1.83 (d, J=6.48 Hz, 2H),1.47-1.72 (m, 14H), 1.21-1.38 (m, 42H), 1.03 (t, J=7.03 Hz, 6H),0.82-0.93 (m, 9H) ppm.

MS (M+1)=757.2, Rt=2.73 min (LC method 13).

Synthesis of Example 130

The following intermediates can be prepared using similar sequences andmethods to those employed for the synthesis of example 129.

Intermediate 130a: 1-((tert-butyldimethylsilyl)oxy)pentadecan-3-ol

TLC: Rf=0.6 (EtOAc:hexanes, 1:9); PMA active.

Intermediate 130b: 1-((tert-butyldimethylsilyl)oxy)pentadecan-3-yl(4-nitrophenyl) carbonate

TLC: Rf=0.9 (EtOAc: hexanes, 0.5:9.5); PMA active.

Intermediate 130c: 1-((tert-butyldimethylsilyl)oxy)pentadecan-3-yl(3-(diethylamino)propyl) carbonate

TLC: Rf=0.5 (MeOH:DCM, 1:9); PMA active.

Intermediate 130d: 3-(diethylamino)propyl (1-hydroxypentadecan-3-yl)carbonate

TLC: Rf=0.4 (MeOH:DCM, 1:9); PMA active.

The following example can be prepared using similar sequences andmethods to those employed for the synthesis of Example 11.

Example 130 4,4-bis(octyloxy)butyl (3-(diethylamino)propyl)pentadecane-1,3-diyl dicarbonate

¹H NMR (400 MHz, CD₂Cl₂) δ=4.74-4.81 (m, 1H), 4.44 (t, J=5.38 Hz, 1H),4.06-4.21 (m, 6H), 3.54 (dt, J=9.29, 6.66 Hz, 2H), 3.38 (dt, J=9.29,6.66 Hz, 2H), 2.51 (q, J=6.81 Hz, 6H), 1.89-1.97 (m, 2H), 1.75-1.82 (m,2H), 1.68-1.74 (m, 2H), 1.49-1.66 (m, 8H), 1.22-1.37 (m, 40H), 1.00 (t,J=7.09 Hz, 6H), 0.85-0.93 (m, 9H) ppm.

MS (M+1)=759.2, Rt=2.62 min (LC method 13).

Synthesis of Example 131 Intermediate 131a:4-((tert-butyldimethylsilyl)oxy)butanal

4-((tert-butyldimethylsilyl)oxy)butan-1-ol and TEA (10.2 mL, 73.4 mmol)were taken into DCM (100 mL). A solution of sulfur trioxide pyridinecomplex (5.84 g, 36.7 mmol) in DMSO (20.8 mL, 294 mmol) was addeddropwise to the alcohol and amine solution in an ice-bath. The resultantmixture was stirred at room temperature for 4 hr. The crude mixture waspartitioned between water and ethyl acetate. The organic layer wascollected, dried, concentrated and purified over silica gel with 10%ethyl acetate/hexane to afford 4.5 g of the desired product as acolorless oil.

TLC: Rf=0.7, Heptane:EtOAc=1:1

Intermediate 131b: 1-((tert-butyldimethylsilyl)oxy)hexadecan-4-ol

Intermediate 131a (4.50 g, 22.2 mmol) was dissolved in 100 mL ofanhydrous THF and cooled to −41° C. 1 M dodecylmagnesium bromide in THF(33.4 mL, 33.4 mmol) was added to the solution. The resulting mixturewas stirred at −41° C. for one hour then warmed up to ambienttemperature for one hour. Sat. NH₄Cl solution was added to quench thereaction. The reaction mixture was extracted with EtOAc (2×100 mL). Thecombined organic phases were dried over MgSO₄, filtered andconcentrated. The residue was purified by silica gel flash columnchromatography with 0-50% EtOAc/Heptane to afford 6.65 g of the desiredproduct as a colorless oil.

¹H NMR (400 MHz, CD₂Cl₂) δ=3.56-3.61 (m, 2H), 3.45-3.53 (m, 1H),1.89-1.98 (m, 1H), 1.48-1.59 (m, 3H), 1.30-1.39 (m, 3H), 1.15-1.27 (m,20H), 0.79-0.89 (m, 12H), −0.01 (s, 6H) ppm.

Intermediate 131c:5-dodecyl-2,2,10,10,11,11-hexamethyl-3,3-diphenyl-4,9-dioxa-3,10-disiladodecane

To intermediate 131b (1.5 g, 4.02 mmol) in anhydrous DCM (15 mL) wasadded imidazole (0.411 g, 6.04 mmol), followed by addition oftert-butyldiphenylchlorosilane (1.24 mL, 4.83 mmol). The reaction wasthen stirred under the presence of nitrogen for 5 h. The reaction wasdiluted with 30 mL of DCM and washed with 50 mL of water. The separatedorganic layer was dried over sodium sulfate, concentrated, and purifiedby flash column chromatography (0-25% EtOAc in heptane) to provide 2.4 gof the desired product as a colorless oil.

¹H NMR (400 MHz, CDCl₃) δ=7.61-7.71 (m, 4H), 7.30-7.45 (m, 6H),3.66-3.77 (m, 1H), 3.37-3.49 (m, 2H), 1.38-1.52 (m, 5H), 1.09-1.34 (m,21H), 1.04 (s, 9H), 0.80-0.90 (m, 12H), 0.00 (s, 6H) ppm.

Intermediate 131d: 4-((tert-butyldiphenylsilyl)oxy)hexadecan-1-ol

To intermediate 131c (2.4 g, 3.93 mmol) in anhydrous EtOH (15 mL) wasadded 10-camphorsulfonic acid (0.411 g, 1.76 mmol). The resultingmixture was stirred at room temperature for 3 h. TLC indicatedcompletion of the reaction. The mixture was diluted with 100 mL of DCMand washed with 50 mL of sat. NaHCO₃. The separated aq. layer wasextracted with an additional 50 mL of DCM. The combined organic layerswere dried over sodium sulfate, concentrated, and purified by flashcolumn chromatography (eluting with 0-50% EtOAc in heptane) to provide1.4 g of the desired product as a colorless oil.

¹H NMR (400 MHz, CDCl₃) δ=7.52-7.64 (m, 4H), 7.23-7.36 (m, 6H),3.60-3.72 (m, 1H), 3.34-3.45 (m, 2H), 1.27-1.50 (m, 7H), 0.92-1.23 (m,20H), 0.90-1.00 (m, 9H), 0.80 (t, J=1.00 Hz, 3H) ppm.

Intermediate 131e:2-(5-((4-((tert-butyldiphenylsilyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyldioctanoate

In a vial, intermediate 1f (1.20 g, 2.82 mmol), intermediate 131d (1.4g, 2.82 mmol), DIPEA (0.984 mL, 5.64 mmol), and DMAP (0.069 g, 0.564mmol) were taken into anhydrous dichloromethane (10 mL). EDC.HCl (1.08g, 5.64 mmol) was added in one portion, and the reaction was stirred atambient temperature overnight. The reaction was then diluted with water(10 mL). The organic layer was separated, and the aqueous layer wasextracted with DCM (10 mL). The combined organic layers were dried overNa₂SO₄, filtered, concentrated and purified by flash columnchromatography (eluting with 0-25% EtOAc:heptane) to provide 2.06 g ofthe desired product as a colorless oil.

¹H NMR (400 MHz, CDCl₃) δ=7.64-7.76 (m, 4H), 7.32-7.47 (m, 6H),4.01-4.12 (m, 4H), 3.94 (t, J=6.66 Hz, 2H), 3.71-3.80 (m, 1H), 2.23-2.37(m, 6H), 1.94-2.06 (m, 1H), 1.58-1.70 (m, 6H), 1.11-1.49 (m, 46H), 1.07(s, 9H), 0.90 (td, J=6.88, 1.77 Hz, 9H) ppm.

Intermediate 131f:2-(5-((4-hydroxyhexadecyl)oxy)-5-oxopentyl)propane-1,3-diyl dioctanoate

To intermediate 131e (2.06 g, 2.27 mmol) in a 100 mL RBF was added 1 MTBAF in THF (11.35 mL, 11.35 mmol). The reaction was stirred at ambienttemperature under the presence of nitrogen overnight. The crude mixturewas diluted with 40 mL of DCM and washed with 40 mL of sat. NaHCO₃. Theaq. layer was extracted with an additional 40 mL of DCM. The organiclayers were combined and dried over sodium sulfate, concentrated andpurified by flash column chromatography (eluting with 0-100% EtOAc inheptane) to afford 800 mg of the desired product as a colorless oil.

¹H NMR (400 MHz, CD₂Cl₂) δ=3.99-4.14 (m, 6H), 3.62 (d, J=5.01 Hz, 1H),2.25-2.38 (m, 6H), 1.94-2.05 (m, 1H), 1.23-1.86 (m, 53H), 0.85-0.98 (m,9H) ppm.

Example 131 2-(5-((4-((1,4-dimethylpiperidine-4-carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyl dioctanoate

Intermediate 131f (70 mg, 0.105 mmol),1,4-dimethylpiperidine-4-carboxylic acid HCl salt (81 mg, 0.419 mmol),DIPEA (0.110 mL, 0.628 mmol), and DMAP (10.2 mg, 0.084 mmol) were takeninto anhydrous dichloromethane (2 mL). EDC.HCl (80 mg, 0.419 mmol) wasadded in one portion, and the reaction was stirred at ambienttemperature overnight. The crude reaction was concentrated under reducedpressure and purified by flash column chromatography (eluting with0-100% EtOAc in heptane,) to provide 62 mg of the desired product as acolorless oil.

¹H NMR (400 MHz, CD₂Cl₂) δ=4.83-4.93 (m, 1H), 3.96-4.07 (m, 6H),2.48-2.58 (m, 2H), 2.28 (t, J=7.58 Hz, 6H), 2.18 (s, 3H), 2.02-2.11 (m,4H), 1.94-2.01 (m, 1H), 1.20-1.68 (m, 54H), 1.16 (s, 3H), 0.82-0.92 (m,9H) ppm.

MS (M+1)=809.2, Rt=2.52 min (LC method 13).

The following example can be prepared using similar sequences andmethods to those employed for the synthesis of example 131.

Example 1322-(5-((4-((1,3-dimethylpyrrolidine-3-carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyldioctanoate

¹H NMR (400 MHz, CD₂Cl₂) δ=4.73-4.82 (m, 1H), 3.89-4.01 (m, 6H), 2.80(d, J=9.17 Hz, 1H), 2.45 (d, J=7.58 Hz, 2H), 2.14-2.33 (m, 11H),1.85-1.94 (m, 1H), 1.40-1.61 (m, 14H), 1.10-1.34 (m, 42H), 0.75-0.87 (m,9H) ppm.

MS (M+1)=795.1, Rt=2.47 min (LC method 13).

Synthesis of Example 133 Intermediate 133a: (2S)-1-tert-butyl2-(1-((7-(octanoyloxy)-6-((octanoyloxy)methyl)heptanoyl)oxy)hexadecan-4-yl)pyrrolidine-1,2-dicarboxylate

Intermediate 133a can be prepared using similar methods to thoseemployed for the synthesis of example 131.

¹H NMR (400 MHz, CD₂Cl₂) δ=4.83-4.94 (m, 1H), 4.22 (dd, J=8.74, 2.87 Hz,1H), 3.89-4.11 (m, 6H), 3.32-3.54 (m, 2H), 2.28 (t, J=7.52 Hz, 6H),2.12-2.23 (m, 1H), 1.81-2.02 (m, 4H), 1.51-1.69 (m, 10H), 1.19-1.48 (m,51H), 0.88 (t, J=6.11 Hz, 9H) ppm.

Example 1332-(5-oxo-5-((4-(((S)-pyrrolidine-2-carbonyl)oxy)hexadecyl)oxy)pentyl)propane-1,3-diyl dioctanoate

4 M HCl in dioxane (1030 μl, 4.16 mmol) was added into intermediate 133a(120 mg, 0.139 mmol) in a vial. The resulting solution was stirred atambient temperature for 1 h. The crude mixture was concentrated underreduced pressure, and redissolved in 5 mL of DCM. The organic layer wasthen washed with 5 mL of 2.0 N Na₂CO₃ solution, dried over sodiumsulfate, concentrated and purified by flash column chromatography(eluting with 0-100% EtOAc in heptane) to provide 48 mg of the desiredproduct.

¹H NMR (400 MHz, CD₂Cl₂) δ=4.85-4.96 (m, 1H), 4.02 (t, J=6.17 Hz, 6H),3.66-3.76 (m, 1H), 3.00-3.10 (m, 1H), 2.82-2.94 (m, 1H), 2.28 (t, J=7.58Hz, 6H), 2.05-2.17 (m, 1H), 1.91-2.04 (m, 1H), 1.67-1.86 (m, 3H),1.50-1.66 (m, 11H), 1.20-1.43 (m, 42H), 0.81-0.92 (m, 9H) ppm.

MS (M+1)=766, Rt=2.23 min (LC method 13).

Synthesis of Example 134 Intermediate 134a:2-(5-((4-(((4-nitrophenoxy)carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyldioctanoate

Intermediate 131f (372 mg, 0.556 mmol) was taken into DCM in a vialcharged with a magnetic stir bar. 4-nitrophenyl carbonochloridate (134mg, 0.667 mmol) was added in one portion. The reaction was capped andplaced under nitrogen, after which pyridine was added dropwise viasyringe. The reaction was allowed to stir at ambient temperatureovernight. The reaction was then diluted with water (10 mL) and DCM (10mL). The organic layer was separated, and the aqueous layer was washedwith DCM (10 mL). The combined organic layers were washed with brine (10mL), dried with Na2SO4, filtered, and concentrated in vacuo. The cruderesidue was purified by flash column chromatography (eluting with 0-25%EtOAc in heptane) to provide 420 mg of the desired product as acolorless oil.

¹H NMR (400 MHz, CD₂Cl₂) δ=8.23-8.31 (m, 2H), 7.34-7.44 (m, 2H),4.81-4.90 (m, 1H), 3.94-4.13 (m, 6H), 2.22-2.35 (m, 6H), 1.92-2.04 (m,1H), 1.54-1.78 (m, 12H), 1.20-1.44 (m, 40H), 0.88 (t, J=7.00 Hz, 9H)ppm.

Example 134 2-(5-((4-(((((S)-1-methylpyrrolidin-3-yl)oxy)carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyldioctanoate

(S)-1-methylpyrrolidin-3-ol (34.0 mg, 0.336 mmol) in a small amount ofanhydrous ACN was added dropwise to intermediate 134a (70 mg, 0.084mmol) in anhydrous ACN (3 mL) in a vial charged with a magnetic stir barunder N₂. Next, pyridine was added via syringe, followed by the additionof DMAP (dissolved in a minimal volume of MeCN) in one portion. Thereaction was allowed to stir at ambient temperature for 3 days. Thecrude mixture was then diluted with 10 mL of water and extracted withDCM (2×10 mL). The combined organic layers were washed with 10 mL ofwater, dried over sodium sulfate, and purified by flash columnchromatography (eluting with 0-100% EtOAc in heptane) to provide 40 mgof the desired product as a clear oil.

¹H NMR (400 MHz, CD₂Cl₂) δ=5.08-5.17 (m, 1H), 4.68-4.79 (m, 1H),3.99-4.13 (m, 6H), 2.70-2.95 (m, 3H), 2.40-2.54 (m, 3H), 2.27-2.38 (m,6H), 1.90-2.08 (m, 2H), 1.52-1.78 (m, 12H), 1.21-1.46 (m, 42H), 0.92 (t,J=7.60 Hz, 9H) ppm.

MS (M+1)=797, Rt=2.48 min (LC method 13).

The following examples can be prepared using similar sequences andmethods to those employed for the synthesis of Example 134.

Example 135 2-(5-((4-(((((R)-1-methylpyrrolidin-3-yl)oxy)carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyl dioctanoate

¹H NMR (400 MHz, CD₂Cl₂) δ=5.08-5.17 (m, 1H), 4.68-4.79 (m, 1H),3.99-4.13 (m, 6H), 2.70-2.95 (m, 3H), 2.40-2.54 (m, 3H), 2.27-2.38 (m,6H), 1.90-2.08 (m, 2H), 1.52-1.78 (m, 12H), 1.21-1.46 (m, 42H), 0.92 (t,J=7.60 Hz, 9H) ppm.

MS (M+1)=797, Rt=2.41 min (LC method 13).

Example 136 2-(5-((4-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyl dioctanoate

¹H NMR (400 MHz, CD₂Cl₂) 6=4.59-4.92 (m, 1H), 3.85-4.12 (m, 8H),2.69-2.88 (m, 2H), 2.22-2.39 (m, 8H), 1.83-2.04 (m, 3H), 1.46-1.78 (m,17H), 1.18-1.42 (m, 40H), 1.02 (t, J=7.15 Hz, 3H), 0.88 (t, J=7.30 Hz,9H) ppm.

MS (M+1)=838, Rt=2.35 min (LC method 13).

Example 1372-(5-((4-((((1-methylpiperidin-4-yl)oxy)carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyldioctanoate

¹H NMR (400 MHz, CD₂Cl₂) δ=4.51-4.92 (m, 2H), 3.93-4.17 (m, 6H),2.56-2.89 (m, 2H), 2.15-2.50 (m, 9H), 1.92-2.07 (m, 3H), 1.73-1.89 (m,2H), 1.43-1.71 (m, 14H), 1.17-1.42 (m, 40H), 0.88 (t, J=7.00 Hz, 9H)ppm.

MS (M+1)=811.4, Rt=2.48 min (LC method 13).

Example 1382-(10-dodecyl-3-ethyl-8,15-dioxo-7,9,14-trioxa-3-azanonadecan-19-yl)propane-1,3-diyldioctanoate

¹H NMR (400 MHz, CD₂Cl₂) δ=4.63-4.74 (m, 1H), 4.14 (td, J=6.51, 2.14 Hz,2H), 3.95-4.08 (m, 6H), 2.48 (br. s., 6H), 2.28 (t, J=7.52 Hz, 6H),1.92-2.03 (m, 1H), 1.77 (br. s, 2H), 1.49-1.68 (m, 12H), 1.19-1.40 (m,40H), 0.99 (br. s., 6H), 0.82-0.93 (m, 9H) ppm.

MS (M+1)=827.2, Rt=2.51 min (LC method 13).

Synthesis of Example 139 Intermediate 139a: 4-(diethylamino)butyl(1-hydroxypentadecan-3-yl) carbonate

Intermediate 139a can be prepared using similar methods to thoseemployed for the synthesis of intermediate 1k.

MS (M+1)=416.7, Rt=1.44 min (LC method 14).

Example 1392-(11-dodecyl-3-ethyl-9,15-dioxo-8,10,14-trioxa-3-azanonadecan-19-yl)propane-1,3-diyldioctanoate

Example 15 can be prepared using similar methods to those employed forthe synthesis of example 1.

¹H NMR (400 MHz, CD₂Cl₂) δ=4.77 (quin, J=6.24 Hz, 1H), 3.95-4.18 (m,8H), 2.38-2.60 (m, 6H), 2.23-2.33 (m, 6H), 1.94-2.03 (m, 1H), 1.90 (q,J=6.44 Hz, 2H), 1.46-1.70 (m, 12H), 1.20-1.42 (m, 40H), 1.01 (t, J=6.66Hz, 6H), 0.82-0.94 (m, 9H) ppm. MS (M+1)=827.3, Rt=2.57 min (LC method13).

Synthesis of Example 140 Intermediate 140a: 3-(1H-imidazol-1-yl)propyl(1-hydroxypentadecan-3-yl) carbonate

Intermediate 140a can be prepared using similar methods to thoseemployed for the synthesis of intermediate 1k.

MS (M+1)=397.6, Rt=0.53 min (LC method 14).

Example 1402-(5-((3-(((3-(1H-imidazol-1-yl)propoxy)carbonyl)oxy)pentadecyl)oxy)-5-oxopentyl)propane-1,3-diyldioctanoate

Example 140 can be prepared using similar methods to those employed forthe synthesis of example 1.

¹H NMR (400 MHz, CD₂Cl₂) δ=8.13 (br. s., 1H), 7.27 (br. s., 1H), 7.15(br. s., 1H), 4.72-4.84 (m, 1H), 4.15-4.25 (m, 4H), 4.03 (dd, J=8.19,5.75 Hz, 6H), 2.25-2.36 (m, 6H), 2.14-2.23 (m, 2H), 1.48-2.00 (m, 11H),1.17-1.41 (m, 40H), 0.82-0.96 (m, 9H) ppm. MS (M+1)=808.2, Rt=2.58 min(LC method 13).

Synthesis of Example 141 Intermediate 141a: 1-hydroxypentadecan-3-yl(3-(piperidin-1-yl)propyl) carbonate

Intermediate 141a can be prepared using similar methods to thoseemployed for the synthesis of intermediate 1k.

MS (M+1)=414.4, Rt=0.53 min (LC method 14).

Example 1412-(5-oxo-5-((3-(((3-(piperidin-1-yl)propoxy)carbonyl)oxy)pentadecyl)oxy)pentyl)propane-1,3-diyldioctanoate

Example 141 can be prepared using similar methods to those employed forthe synthesis of example 1.

¹H NMR (400 MHz, CD₂Cl₂) δ=4.77 (quin, J=6.24 Hz, 1H), 3.95-4.18 (m,8H), 2.20-2.48 (m, 12H), 1.95-2.03 (m, 1H), 1.90 (m, J=6.40, 6.40, 6.40Hz, 4H), 1.52-1.69 (m, 12H), 1.19-1.50 (m, 42H), 0.82-0.95 (m, 9H) ppm.

MS (M+1)=825.2, Rt=2.52 min (LC method 13).

Synthesis of Example 142 Intermediate 142a: 3-(benzyloxy)pentadecan-1-ol

To a solution of intermediate 1h (500 mg, 1.39 mmol) in anhydrous DMF (5mL) at 0° under the presence of nitrogen was added 60% NaH in mineraloil (84 mg, 2.091 mmol). The resulting mixture was stirred at 0° C. for30 min before benzyl bromide (0.249 mL, 2.091 mmol) was added. Thereaction was allowed to warm to ambient temperature and stirredovernight. The reaction was quenched with 5 mL of sat. NH₄Cl and 10 mLof water, and diluted with 15 mL of DCM. The separated aq. layer wasextracted with an additional 10 mL of DCM. The separated organic layerswere combined and washed with 10 mL of water, dried over sodium sulfate,concentrated and purified by flash column chromatography (eluting with0-50% EtOAc in heptane) to provide 450 mg of impure desired product.This material was used in the next step without further purification.

This compound (450 mg, 1.00 mmol) was then taken into MeOH in a vialcharged with a magnetic stir bar. CAN (1.20 g, 2.20 mmol) was added inone portion. The reaction was allowed to stir at ambient temperatureunder nitrogen for 1 hour, after which the desired product was the majorproduct observed by LCMS. The reaction was quenched with sat. NaHCO₃ andthe organics were extracted with DCM (×3). The combined organic layerswere washed with brine, dried with Na₂SO₄, and purified by flash columnchromatography (eluting with 0-50% EtOAc in heptane) to provide 30 mg ofthe desired product.

¹H NMR (400 MHz, CD₂Cl₂) δ=7.17-7.34 (m, 5H), 4.43 (s, 2H), 3.48-3.72(m, 3H), 1.52-1.71 (m, 2H), 1.30-1.41 (m, 3H), 1.19 (br. s, 20H), 0.81(t, J=1.00 Hz, 3H) ppm.

Intermediate 142b:2-(5-((3-(benzyloxy)pentadecyl)oxy)-5-oxopentyl)propane-1,3-diyldioctanoate

Intermediate 142a (130 mg, 0.389 mmol), intermediate 1f (250 mg, 0.583mmol), DMAP (18.99 mg, 0.155 mmol), and DIPEA (0.136 mL, 0.777 mmol)were taken into anhydrous dichloromethane (4 mL) in a vial. EDC.HCl (149mg, 0.777 mmol) was added in one portion, and the reaction was stirredat ambient temperature over the weekend. The reaction was then dilutedwith H₂O (10 mL) and DCM (10 mL). The organic layer was separated, andthe aqueous layer was washed with DCM (100 mL). The combined organiclayers were washed with brine (5 mL), dried with Na₂SO₄, filtered, andconcentrated in vacuo to provide a crude orange oil. The crude oil waspurified by flash column chromatography (eluting with 0-100%EtOAc:heptane) to provide 163 mg of the desired product as a yellow oil.

¹H NMR (400 MHz, CD₂Cl₂)) δ=7.24-7.39 (m, 5H), 5.00 (quin, J=1.00 Hz,1H), 4.45 (s, 2H), 3.95-4.10 (m, 4H), 3.47 (td, J=6.42, 4.52 Hz, 2H),2.20-2.31 (m, 6H), 1.92-2.01 (m, 1H), 1.79-1.87 (m, 2H), 1.51-1.65 (m,8H), 1.19-1.42 (m, 40H), 0.88 (t, J=1.00 Hz, 9H) ppm.

Intermediate 142c:2-(5-((1-hydroxypentadecan-3-yl)oxy)-5-oxopentyl)propane-1,3-diyldioctanoate

A vial containing intermediate 142b (163 mg, 0.218 mmol) and a magneticstir bar was evacuated and flushed with N₂ (×3). Next, 5% Pd/C (ca 50%H2O) was added in one portion, and the N₂ flush was repeated. EtOH wasthen added in one portion via syringe down the sides of the flask. Theflask was placed under atmospheric H₂ pressure via a balloon and allowedto stir at room temperature for 1 h. The reaction was filtered and thefiltrate was concentrated under reduced pressure, which afforded 123 mgof the desired product as a colorless oil.

¹H NMR (400 MHz, CD₂Cl₂) δ=4.95-5.03 (m, 1H), 3.98-4.08 (m, 4H),3.44-3.61 (m, 2H), 2.26-2.35 (m, 6H), 1.94-2.02 (m, 1H), 1.73-1.84 (m,1H), 1.51-1.69 (m, 10H), 1.34-1.41 (m, 4H), 1.20-1.33 (m, 36H),0.84-0.91 (m, 9H) ppm.

Intermediate 142d:2-(5-((1-(((4-nitrophenoxy)carbonyl)oxy)pentadecan-3-yl)oxy)-5-oxopentyl)propane-1,3-diyldioctanoate

Intermediate 142c (120 mg, 0.183 mmol) was taken into DCM (3 mL) in avial charged with a magnetic stir bar. 4-nitrophenyl carbonochloridatewas added in one portion. The reaction was capped and placed undernitrogen, after which pyridine was added dropwise via syringe. Thereaction was allowed to stir at ambient temperature overnight. Thereaction was diluted with H₂O (5 mL) and DCM (5 mL). The organic layerwas separated, and the aqueous layer was washed with DCM (5 mL). Thecombined organic layers were washed with brine (10 mL), dried withNa₂SO₄, concentrated, and purified by flash column chromatography(eluting with 0-50% EtOAc in heptane) to provide 126 mg of the desiredproduct as a white wet solid (contained minor aromatic impurities).

¹H NMR (400 MHz, CD₂Cl₂) δ=8.22-8.32 (m, 2H), 7.33-7.44 (m, 2H),4.97-5.08 (m, 1H), 4.25-4.36 (m, 2H), 3.94-4.09 (m, 4H), 2.21-2.37 (m,6H), 1.88-2.07 (m, 3H), 1.49-1.68 (m, 8H), 1.19-1.43 (m, 40H), 0.88 (t,J=1.00 Hz, 9H) ppm.

Example 1422-(12-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azaoctadecan-18-yl)propane-1,3-diyldioctanoate

3-(diethylamino)propan-1-ol in 1 mL of anhydrous ACN was added dropwiseto a solution of Intermediate 142d (50 mg, 0.061 mmol) in ACN (1 mL) ina vial charged with a magnetic stir bar under N₂. Next, pyridine wasadded via syringe, followed by the addition of DMAP (1.49 mg, 0.012mmol) (dissolved in 0.1 mL of MeCN) in one portion. The reaction wasallowed to stir at ambient temperature overnight. The crude mixture wasthen diluted with 10 mL of water and extracted with DCM (2×10 mL). Thecombined organic layers were dried over sodium sulfate, concentrated,and purified by flash column chromatography (eluting with 0-100% EtOAcin heptane) to provide 28 mg of the desired product as a colorless oil.

¹H NMR (400 MHz, CD₂Cl₂) δ=4.90-5.00 (m, 1H), 4.09-4.17 (m, 4H),3.94-4.08 (m, 4H), 2.47 (q, J=7.30 Hz, 6H), 2.28 (t, J=7.52 Hz, 6H),1.71-2.02 (m, 5H), 1.49-1.66 (m, 8H), 1.34-1.41 (m, 4H), 1.19-1.34 (m,36H), 0.98 (t, J=7.09 Hz, 6H), 0.84-0.93 (m, 9H) ppm.

MS (M+1)=813.2, Rt=2.61 min (LC method 13).

siRNA Lipid Formulations

The lipid nanoparticles (LNPs) were formed by mixing equal volumes oflipids dissolved in alcohol with siRNA dissolved in a citrate buffer byan impinging jet process. The lipid solution contains a cationic lipidcompound of the invention, a helper lipid (cholesterol), an optionalneutral lipid (DSPC) and a PEG (PEG) lipid at a concentration of 8-16mg/mL with a target of 12 mg/mL in an alcohol. The siRNA to total lipidratio is approximately 0.05 (wt/wt). Where a LNP formulation containsfour lipid components, the molar ratios of the lipids ranges from 20 to70 mole percent for the cationic lipid with a target of 40-60, the molepercent of helper lipid ranges from 20 to 70 with a target of 30 to 50,the mole percent of neutral lipid ranges from 0-30, the mole percent ofPEG lipid has a range from 1 to 6 with a target of 2 to 5. Theconcentration of siRNA solution ranges from 0.7 to 1.0 mg/mL with atarget of 0.8 to 0.9 mg/mL in a sodium citrate: sodium chloride bufferpH 4-6, with a target of 4.5-5.5. The LNPs are formed by mixing equalvolumes of lipid solution in ethanol with siRNA dissolved in a citratebuffer by an impinging jet process through a mixing device with IDranging from 0.25 to 2.0 mm at a flow rate from 10 to 640 mL/min. Themixed LNP solution is held at room temperature for 0-24 hrs prior to adilution step. The solution is then concentrated and diafiltered withsuitable buffer by ultrafiltration or dialysis process using membraneswith a MW cutoff from 30 to 500 KD. The final product is sterilefiltered and stored at 4° C.

siRNA's

The siRNA used in the lipid nanoparticles described was made up ofdouble stranded siRNA sequences specific to a target mRNA sequence.

1. FVII siRNA duplex sequence

(SEQ ID NO: 1) 5′ UUu AAU UGA AAC cAA GAc Auu 3′ (SEQ ID NO: 2) 5′uGu cuu GGu uuc AAu uAA Auu 3′

2. PLK1-424 siRNA duplex sequence

(SEQ ID NO: 3) 5′ UAU UUA AgG AGG GUG AuC Uuu 3′ (SEQ ID NO: 4) 5′AGA Uca cCC Ucc uuA AAU auu 3′

The following abbreviations are used in these sequences:

-   -   A=adenosine    -   U=uridine    -   G=guanosine    -   C=cytosine    -   a=2′-O-methyl-adenosine    -   u=2′-O-methyl-uridine    -   g=2′-O-methyl-guanosine    -   c=2′-O-methyl-cytosine

Plasmid's

pcDNA3.1(-)Neo from LifeTechnologies Cat# V795-20 (SEQ ID NO: 5)GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACGGGCCCTCTAGACTCGAGCGGCCGCCACTGTGCTGGATATCTGCAGAATTCCACCACACTGGACTAGTGGATCCGAGCTCGGTACCAAGCTTAAGTTTAAACCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCpGEM-T7o-TEV-hLeptin-GAopt-2xhBG-120A sequence (SEQ ID NO: 6)GATCCGGAGGCCGGAGAATTGTAATACGACTCACTATAGGGAGACGCGTGTTAAATAACAAATCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAGCCGCCACCATGCACTGGGGAACCCTGTGCGGATTCCTGTGGCTGTGGCCCTACCTGTTCTATGTGCAAGCCGTGCCCATCCAGAAGGTGCAGGACGACACCAAGACCCTGATCAAGACCATCGTGACCCGGATCAACGACATCAGCCACACCCAGAGCGTGTCCAGCAAGCAGAAAGTGACCGGCCTGGACTTCATCCCCGGCCTGCACCCTATCCTGACCCTGTCCAAGATGGACCAGACCCTGGCCGTGTACCAGCAGATCCTGACCAGCATGCCCAGCCGGAACGTGATCCAGATCAGCAACGACCTGGAAAACCTGCGGGACCTGCTGCACGTGCTGGCCTTCAGCAAGAGCTGCCATCTGCCTTGGGCCAGCGGCCTGGAAACCCTGGATTCTCTGGGCGGAGTGCTGGAAGCCAGCGGCTACTCTACAGAGGTGGTGGCCCTGAGCAGACTGCAGGGCAGCCTGCAGGATATGCTGTGGCAGCTGGATCTGAGCCCCGGCTGCTAATAGCGGACCGGCGATAGATGAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCGGCCGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAGAGCAAGCTTTCGATAGCGCTGTTCGTAGAAAAAAAAGAAGTAAATAATTACTACTTGCCATATAGACTAAATAGCTGCGCNTAATACATCTACACTTTCTANNATTGACAAGTGATACGTTGCAAAAGGAGCAACACCCCACAGACTCGATGACTGCGCAGTCATACAGTGAAATTGCCCTAATGTCTTACCTCTGAAAGGGCTAAACGAAAGTAGAGCACTATTCCGCGTAGCTATTTAGTGCGATCTTTTAGAAATATCAGCCCAGAGAGCTGGGCTGATAAATATTTTATCCGACAAGACGAATTTTGCTCAAATGAGTTAAAACGATGCTACCACTATCTGCTGCTTTTACGAGATCAGCCCACCATTGCATCATCGGACGACGTTGCTCAAGATAATCACTGCGGTTATAAGCGCGACGCACCTCATTTTTGTCTACATGAGCAAGCGCTGCTTCAATGACATCAGGTGGAAATCCTTCCTCATTGAGTGCCGTACTGGCGATAGAACGCAAGCCGTGTGAAACAAGTACACCTCCTAAGCCAGCACGCTTGAGTGCTGCATTCACTGTTTGGCTATTCATTGGTTGGTTGGGCTTGATACGGCTAGGAAAGATAAATTCTCGGCCACCACTGAGAGGCTTCATCATTTCCAGAATAGCAAGAGCCCCATCAGATAGTGGAACCGTATGGTCCCGGTTCATCTTCATTCGAGCTGCAGGAATTTTCCATTCGCTAGCATTGAAATCGATCTCATCCCATCGAGCCTCAGCAGCTTCGGCAGGGCGGGTGATGGTTAGAAGTTGCCACATGAACAGGCATCTTGTGGACATGCTGATACTTGCCGTACGCATGGTGTGCATTAGCTGCGGAAGTTGATCCGGCCGGATGCTTGGCATGTTTTTCTTTTGCGGTTTCTCGAAAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTATTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCGATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGGCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAAATTCGAGCTCGGTACCCGGGmRNA'sBrief Description of mRNA Transcription Protocol

A circular plasmid DNA template is constructed that contains a mRNAtranscription cassette consisting of the following features: a consensusT7 bacteriophage DNA-dependent RNA polymerase promoter, a 5′untranslated region (UTR), a Kozak sequence, and open reading frame, a3′ UTR, and a 120 nucleotide long polyadenosine (polyA120) tail (SEQ IDNO: 13). The plasmid DNA template is propagated in E. coli, isolated,and linearized by restriction enzyme digest immediately 3′ of thepoly120 tail. The plasmid DNA is combined with T7 RNA polymerase,ribonucleotide triphosphates, RNase inhibitor, pyrophosphatase enzyme,dithiothreitol, spermidine, and enzyme reaction buffer and is incubatedfor 1 hour at 37° C. DNase I enzyme is added to digest the plasmid DNAtemplate and is incubated for 0.5 hours at 37° C. mRNA is isolated bysequential precipitation with lithium chloride, washing of the pellet in70% ethanol, resuspension of the mRNA pellet in water, re-precipitationwith isopropanol and sodium acetate, and washing of the pellet again in70% ethanol. The final mRNA pellet is resuspended in water.

Reagent Concentration Notes Nuclease-free water Remaining volumeTris-HCl pH 8.0 (mM) 40 MgCl₂ (mM) 20 ATP, CTP, GTP, UTP 4 (mM)Pseudouridine (mM) 4 To make 100% PsU mRNA, do not include UTP inreaction. To make 100% unmodified mRNA, do not include PsU in reactionDTT (mM) 10 Spermidine (mM) 2 Linearized plasmid 0.05 DNA (ug/ul)Pyrophosphatase 0.004 (U/ul) RNase inhibitor (U/ul) 1 T7 RNA polymerase5 (U/ul) DNase I (U/ul) 0.04

TEV-hLeptin-GAopt-2xhBG-120A (SEQ ID NO:7) Sequence Features: TobaccoEtch Virus (TEV) 5′ UTR: 14-154

Optimal Kozak sequence: 155-163Human leptin encoding amino acids 1-167 of Protein Accession #NP_000221, sequencecodon optimized by GeneArt: 164-6642 stop codons: 665-6702 copies of human beta-globin 3′UTR: 689-954120 nucleotide polyA tail: 961-1080

GGGAGACGCGUGUUAAAUAACAAAUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGCCGCCACCAUGCACUGGGGAACCCUGUGCGGAUUCCUGUGGCUGUGGCCCUACCUGUUCUAUGUGCAAGCCGUGCCCAUCCAGAAGGUGCAGGACGACACCAAGACCCUGAUCAAGACCAUCGUGACCCGGAUCAACGACAUCAGCCACACCCAGAGCGUGUCCAGCAAGCAGAAAGUGACCGGCCUGGACUUCAUCCCCGGCCUGCACCCUAUCCUGACCCUGUCCAAGAUGGACCAGACCCUGGCCGUGUACCAGCAGAUCCUGACCAGCAUGCCCAGCCGGAACGUGAUCCAGAUCAGCAACGACCUGGAAAACCUGCGGGACCUGCUGCACGUGCUGGCCUUCAGCAAGAGCUGCCAUCUGCCUUGGGCCAGCGGCCUGGAAACCCUGGAUUCUCUGGGCGGAGUGCUGGAAGCCAGCGGCUACUCUACAGAGGUGGUGGCCCUGAGCAGACUGCAGGGCAGCCUGCAGGAUAUGCUGUGGCAGCUGGAUCUGAGCCCCGGCUGCUAAUAGCGGACCGGCGAUAGAUGAAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCGGCCGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

TEV-mEpo(Ncol)-2xhBG-120A (SEQ ID NO:8) Sequence Features: Tobacco EtchVirus (TEV) 5′ UTR: 14-154

Optimal Kozak sequence: 155-163Mouse erythropoietin encoding amino acids 1-191 of Protein Accession #NP_031968,sequence codon optimized by GeneArt: 164-739Stop codons: 740-7422 copies of human beta-globin 3′UTR: 743-1008120 nucleotide polyA tail: 1009-1128

GGGAGACGCGUGUUAAAUAACAAAUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGCCGCCACCAUGGGCGUGCCCGAAAGACCUACACUCCUGCUGCUGCUGUCACUGCUGCUGAUCCCUCUGGGCCUGCCUGUGCUGUGUGCCCCCCCUAGACUGAUCUGCGACAGCAGAGUGCUGGAACGGUACAUCCUGGAAGCCAAAGAGGCCGAGAACGUGACGAUGGGAUGUGCCGAGGGCCCCAGACUGAGCGAGAACAUCACCGUGCCCGACACCAAAGUGAACUUCUACGCCUGGAAGCGGAUGGAAGUGGAAGAACAGGCCAUCGAAGUGUGGCAGGGCCUGAGCCUGCUGAGCGAGGCUAUUCUGCAGGCACAGGCUCUGCUGGCCAACAGCAGCCAGCCUCCUGAGACACUGCAGCUGCACAUCGACAAGGCCAUCAGCGGCCUGAGAAGCCUGACCUCCCUGCUGAGGGUGCUGGGAGCCCAGAAAGAACUGAUGAGCCCCCCUGACACCACCCCCCCUGCUCCUCUGAGAACUCUGACCGUGGACACCUUCUGCAAGCUGUUCCGGGUGUACGCCAACUUCCUGCGGGGCAAGCUGAAGCUGUACACCGGCGAAGUGUGCAGACGGGGCGACAGAUGAAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

TEV-hFIX-GAopt-2xhBG-120A (SEQ ID NO:9) Sequence Features: Tobacco EtchVirus (TEV) 5′ UTR: 14-154

Optimal Kozak sequence: 155-163Human factor IX encoding amino acids 1-461 of Protein Accession #NP_000124, sequencecodon optimized by GeneArt: 164-19622 stop codons: 1547-15522 copies of human beta-globin 3′UTR: 1571-1836120 nucleotide polyA tail: 1843-1962

GGGAGACGCGUGUUAAAUAACAAAUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGCCGCCACCAUGCAGCGCGUGAACAUGAUUAUGGCCGAGAGCCCUGGCCUGAUCACCAUCUGCCUGCUGGGCUACCUGCUGAGCGCCGAGUGCACCGUGUUUCUGGACCACGAGAACGCCAACAAGAUCCUGAACCGGCCCAAGCGGUACAACAGCGGCAAGCUGGAAGAGUUCGUGCAGGGCAACCUGGAACGCGAGUGCAUGGAAGAGAAGUGCAGCUUCGAAGAGGCCAGAGAGGUGUUCGAGAACACCGAGCGGACCACCGAGUUCUGGAAGCAGUACGUGGACGGCGACCAGUGCGAGAGCAACCCCUGUCUGAAUGGCGGCAGCUGCAAGGACGACAUCAACAGCUACGAGUGCUGGUGCCCCUUCGGCUUCGAGGGCAAGAACUGCGAGCUGGACGUGACCUGCAACAUCAAGAACGGCAGAUGCGAGCAGUUCUGCAAGAACAGCGCCGACAACAAGGUCGUGUGCUCCUGCACCGAGGGCUACAGACUGGCCGAGAACCAGAAGUCCUGCGAGCCCGCCGUGCCUUUCCCAUGUGGAAGAGUGUCCGUGUCCCAGACCAGCAAGCUGACCAGAGCCGAGACAGUGUUCCCCGACGUGGACUACGUGAACAGCACCGAGGCCGAGACAAUCCUGGACAACAUCACCCAGAGCACCCAGUCCUUCAACGACUUCACCAGAGUCGUGGGCGGCGAGGAUGCCAAGCCUGGACAGUUCCCGUGGCAGGUGGUGCUGAACGGAAAGGUGGACGCCUUUUGCGGCGGCAGCAUCGUGAACGAGAAGUGGAUCGUGACAGCCGCCCACUGCGUGGAAACCGGCGUGAAGAUUACAGUGGUGGCCGGCGAGCACAACAUCGAGGAAACCGAGCACACAGAGCAGAAACGGAACGUGAUCAGAAUCAUCCCCCACCACAACUACAACGCCGCCAUCAACAAGUACAACCACGAUAUCGCCCUGCUGGAACUGGACGAGCCCCUGGUGCUGAAUAGCUACGUGACCCCCAUCUGUAUCGCCGACAAAGAGUACACCAACAUCUUUCUGAAGUUCGGCAGCGGCUACGUGUCCGGCUGGGGCAGAGUGUUUCACAAGGGCAGAUCCGCUCUGGUGCUGCAGUACCUGAGAGUGCCUCUGGUGGACCGGGCCACCUGUCUGAGAAGCACCAAGUUCACCAUCUACAACAACAUGUUCUGCGCCGGCUUUCACGAGGGCGGCAGAGAUAGCUGUCAGGGCGAUUCUGGCGGCCCUCACGUGACAGAGGUGGAAGGCACCAGCUUUCUGACCGGCAUCAUCAGCUGGGGCGAGGAAUGCGCCAUGAAGGGGAAGUACGGCAUCUACACCAAGGUGUCCAGAUACGUGAACUGGAUCAAAGAAAAGACCAAGCUGACAUAAUGACGGACCGGCGAUAGAUGAAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCGGCCGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA

TEV-Fluc(sapl)-2xhBG-120A (SEQ ID NO: 10) Sequence Features: TobaccoEtch Virus (TEV) 5′ UTR: 14-154

Optimal Kozak sequence: 155-163Sequence encoding polypeptide 99% identical (545/550 aa) to Firefly(Photinus pyralis)luciferase of Protein Accession # P08659: 164-18131 stop codon: 1814-18162 copies of human beta-globin 3′UTR: 1835-2100120 nucleotide polyA tail: 2107-2226

GGGAGACGCGUGUUAAAUAACAAAUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGCCGCCACCAUGGAAGACGCCAAAAACAUAAAGAAAGGCCCGGCGCCAUUCUAUCCGCUGGAAGAUGGAACCGCUGGAGAGCAACUGCAUAAGGCUAUGAAGAGAUACGCCCUGGUUCCUGGAACAAUUGCUUUUACAGAUGCACAUAUCGAGGUGGACAUCACUUACGCUGAGUACUUCGAAAUGUCCGUUCGGUUGGCAGAAGCUAUGAAACGAUAUGGGCUGAAUACAAAUCACAGAAUCGUCGUAUGCAGUGAAAACUCUCUUCAAUUCUUUAUGCCGGUGUUGGGCGCGUUAUUUAUCGGAGUUGCAGUUGCGCCCGCGAACGACAUUUAUAAUGAACGUGAAUUGCUCAACAGUAUGGGCAUUUCGCAGCCUACCGUGGUGUUCGUUUCCAAAAAGGGGUUGCAAAAAAUUUUGAACGUGCAAAAAAAGCUCCCAAUCAUCCAAAAAAUUAUUAUCAUGGAUUCUAAAACGGAUUACCAGGGAUUUCAGUCGAUGUACACGUUCGUCACAUCUCAUCUACCUCCCGGUUUUAAUGAAUACGAUUUUGUGCCAGAGUCCUUCGAUAGGGACAAGACAAUUGCACUGAUCAUGAACUCCUCUGGAUCUACUGGUCUGCCUAAAGGUGUCGCUCUGCCUCAUAGAACUGCCUGCGUGAGAUUCUCGCAUGCCAGAGAUCCUAUUUUUGGCAAUCAAAUCAUUCCGGAUACUGCGAUUUUAAGUGUUGUUCCAUUCCAUCACGGUUUUGGAAUGUUUACUACACUCGGAUAUUUGAUAUGUGGAUUUCGAGUCGUCUUAAUGUAUAGAUUUGAAGAGGAGCUGUUUCUGAGGAGCCUUCAGGAUUACAAGAUUCAAAGUGCGCUGCUGGUGCCAACCCUAUUCUCCUUCUUCGCCAAAAGCACUCUGAUUGACAAAUACGAUUUAUCUAAUUUACACGAAAUUGCUUCUGGUGGCGCUCCCCUCUCUAAGGAAGUCGGGGAAGCGGUUGCCAAGAGGUUCCAUCUGCCAGGUAUCAGGCAAGGAUAUGGGCUCACUGAGACUACAUCAGCUAUUCUGAUUACACCCGAGGGGGAUGAUAAACCGGGCGCGGUCGGUAAAGUUGUUCCAUUUUUUGAAGCGAAGGUUGUGGAUCUGGAUACCGGGAAAACGCUGGGCGUUAAUCAAAGAGGCGAACUGUGUGUGAGAGGUCCUAUGAUUAUGUCCGGUUAUGUAAACAAUCCGGAAGCGACCAACGCCUUGAUUGACAAGGAUGGAUGGCUACAUUCUGGAGACAUAGCUUACUGGGACGAAGACGAACACUUCUUCAUCGUUGACCGCCUGAAGUCUCUGAUUAAGUACAAAGGCUAUCAGGUGGCUCCCGCUGAAUUGGAAUCCAUCUUGCUCCAACACCCCAACAUCUUCGACGCAGGUGUCGCAGGUCUUCCCGACGAUGACGCCGGUGAACUUCCCGCCGCCGUUGUUGUUUUGGAGCACGGAAAGACGAUGACGGAAAAAGAGAUCGUGGAUUACGUCGCCAGUCAAGUAACAACCGCGAAAAAGUUGCGCGGAGGAGUUGUGUUUGUGGACGAAGUACCGAAAGGUCUUACCGGAAAACUCGACGCAAGAAAAAUCAGAGAGAUCCUCAUAAAGGCCAAGAAGGGCGGAAAGAUCGCCGUGUGACGGACCGGCGAUAGAUGAAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCGGCCGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

Gtx7-Gluc-2xSinV-120A (SEQ ID NO: 11) Sequence Features: 5′ UTR: 14-127

Kozak sequence: 128-133Sequence encoding polypeptide identical to Gaussia princeps luciferaseof Protein Accession

# AAG54095: 134-688

1 stop codon: 689-6912 copies of Sindbis Virus 3′UTR: 699-882120 nucleotide polyA tail: 891-1010

GGGAGACGCGUGUUUCUGACAUCCGGCGGAAUUCUGACAUCCGGCGGAAUUCUGACAUCCGGCGGAAUUCUGACAUCCGGCGGAAUUCUGACAUCCGGCGGAAGACUCACAACCCCAGAAACAGACAGCCACCAUGGGAGUCAAAGUUCUGUUUGCCCUGAUCUGCAUCGCUGUGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACUUCAACAUCGUGGCCGUGGCCAGCAACUUCGCGACCACGGAUCUCGAUGCUGACCGCGGGAAGUUGCCCGGCAAGAAGCUGCCGCUGGAGGUGCUCAAAGAGAUGGAAGCCAAUGCCCGGAAAGCUGGCUGCACCAGGGGCUGUCUGAUCUGCCUGUCCCACAUCAAGUGCACGCCCAAGAUGAAGAAGUUCAUCCCAGGACGCUGCCACACCUACGAAGGCGACAAAGAGUCCGCACAGGGCGGCAUAGGCGAGGCGAUCGUCGACAUUCCUGAGAUUCCUGGGUUCAAGGACUUGGAGCCAAUGGAGCAGUUCAUCGCACAGGUCGAUCUGUGUGUGGACUGCACAACUGGCUGCCUCAAAGGGCUUGCCAACGUGCAGUGUUCUGACCUGCUCAAGAAGUGGCUGCCGCAACGCUGUGCGACCUUUGCCAGCAAGAUCCAGGGCCAGGUGGACAAGAUCAAGGGGGCCGGUGGUGACUAACGGACCGAAAACUCAAUGUAUUUCUGAGGAAGCGUGGUGCAUAAUGCCACGCAGUGUCUACAUAAUCAAUUUAUUAUUUUCUUUUAUUUUAUUCACAUAAAAACUCAAUGUAUUUCUGAGGAAGCGUGGUGCAUAAUGCCACGCAGUGUCUACAUAAUCAAUUUAUUAUUUUCUUUUAUUUUAUUCACAUAGCGGCCGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAA

Biological Evaluation

Packaging of mRNA

All equipment and disposable supplies are certified free of RNaseactivity by the manufacturer or rendered RNase free by use of theRNaseZap reagent (LifeTechnologies). mRNA is encapsulated at a cationiclipid amine to mRNA phosphate (N:P) molar ratio of 4:1. Lipids (cationiclipid, DSPC, cholesterol and lipidated PEG or stealth lipid) aredissolved in ethanol. The molar ratios are 40:10:38:2, respectively. Themixture is sonicated briefly, then gently agitated for 5 minutes andthen maintained at 37° C. until use. mRNA is exchanged into citratebuffer pH 5.8-6.0 by use of Amicon Ultra-15 centrifugal concentrators,and the final concentration is adjusted to 0.5 mg/ml and held at 37° C.until use. An equal volume of lipids in ethanol, mRNA in citrate buffer,and citrate buffer alone are drawn into disposable syringes. Tubingleading from syringes containing lipids and mRNA are attached to the Tjunction, and tubing leading from the syringe containing citrate bufferalone is paired with the tubing exiting the T-junction over a collectionvessel containing a stir bar on an active stir plate. Syringes areplaced in a syringe pump set to expel contents at a flow rate of 1 mlper minute.

The pump is activated, and the collected mRNA in lipid nanoparticles istransferred to SnakeSkin dialysis tubing (10,000 MWCO, ThermoScientific). Material is dialyzed against RNAse- and pyrogen-free 1×phosphate buffered saline overnight at 4° C.

Packaging of siRNA

The lipid nanoparticles (LNPs) were formed by mixing equal volumes oflipids dissolved in alcohol with siRNA dissolved in a citrate buffer byan impinging jet process. The lipid solution contains a cationic lipidcompound of the invention, a helper lipid (cholesterol), an optionalneutral lipid (DSPC) and a stealth lipid (5010, S024, S027, or S031) ata concentration of 8-16 mg/mL with a target of 12 mg/mL in an alcohol.The siRNA to total lipid ratio is approximately 0.05 (wt/wt). Where aLNP formulation contains four lipid components, the molar ratios of thelipids ranges from 20 to 70 mole percent for the cationic lipid with atarget of 40-60, the mole percent of helper lipid ranges from 20 to 70with a target of 30 to 50, the mole percent of neutral lipid ranges from0-30, the mole percent of PEG lipid has a range from 1 to 6 with atarget of 2 to 5. The concentration of siRNA solution ranges from 0.7 to1.0 mg/mL with a target of 0.8 to 0.9 mg/mL in a sodium citrate: sodiumchloride buffer pH 4-6, with a target of 4.5-5.5. The LNPs are formed bymixing equal volumes of lipid solution in ethanol with siRNA dissolvedin a citrate buffer by an impinging jet process through a mixing devicewith ID ranging from 0.25 to 2.0 mm at a flow rate from 10 to 640mL/min. The mixed LNP solution is held at room temperature for 0-24 hrsprior to a dilution step. The solution is then concentrated anddiafiltered with suitable buffer by ultrafiltration process usingmembranes with a MW cutoff from 30 to 500 KD. The final product issterile filtered and stored at 4° C.

Measurement of mRNA Encapsulation

Percent encapsulation of mRNA in lipid nanoparticles is determined usingthe Quant-iT Ribogreen RNA Assay kit (Life Technologies). The LNP-mRNAsuspension is assayed in buffer (mRNA outside the particle), and bufferplus Triton X-100 detergent (total mRNA). The difference calculated isthe mRNA inside the particle. Prepare a 1000 ng/mL stock from the RNAprovided in the kit and use this to generate a standard curve (0 ng/ml,15.63-1000 ng/ml) in TE and TE+0.75% Triton X-100. Prepare LNP-mRNAsamples in TE buffer and TE buffer +0.75% Triton X-100 with appropriatedilution so that reading is in the range of standard curve (400-2,000fold). In a 384-well plate (Costar non-treated #3573) add 0.04 ml ofstandard (in duplicate) or sample (in triplicate) per well. DiluteRibogreen reagent 240-fold in TE buffer and add 0.06 ml per well. Mixcontents of wells and measure fluorescence (excitation=480 nm,emission=520 nm). Subtract background values (no RNA) from standard andtest sample values and determine the concentrations of RNA in thesamples using the standard curves. Determine the percent encapsulationof the sample by dividing the difference in concentrations betweensample+triton and sample in buffer alone by the sample+tritonconcentration.

Measurement of siRNA Encapsulation

SYBR Gold flourescence reagent is used for the determination of siRNAencapsulation in the DLP's. DLP's with and without triton x-100 are usedto determine the free siRNA and total siRNA amounts. DLP's samples withand without triton X-100 were excited at 485 nm and flourescenceemission is measured at 530 nm. Encapsulation efficiency is calculatedbased on the following formula:

Encapsulation efficiency:[(free siRNA concentration−total siRNAconcentration)/(total siRNA concentration)]×100%

Encapsulation Data

TABLE 2 In-vitro encapsulation data for mRNA and siRNA % encapsulationmRNA siRNA Example Leptin FVII 1 97.1 NA 4 NA 88.6 6 24.0 91.7 9 96.691.0 10 88.4 89.9 11 98.2 89.7 12 NA 91.7 13 81.1 90.3 14 99.4 88.0 1598.9 91.0 16 97.7 88.9 17 84.6 88.3 18 97.1 91.2 19 NA 84.5 20 NA 76.721 98.2 91.2 22 99.2 91.7 23 98.0 92.0 24 NA 86.8 25 NA 87.1 26 NA 89.827 NA 86.6 29 NA 86.1 43 NA 86.8 44 NA 79.8 45 88.2 86.4 46 NA 85.2 48NA 89.3 49 NA 88.4 50 NA 85.3 51 NA 89.2 52 NA 84.6 53 NA 85.6 54 NA88.6 58 90.2 NA 59 NA 90.2 60 NA 82.4 61 NA 85.5 62 NA 75.2 63 NA 85.664 93.0 NA 65 NA 39.5 66 NA 83.4 70 98.3 80.0 71 NA 86.7 72 NA 62.5 80NA 85.2 81 NA 48.8 82 81.9 NA 83 48.9 NA 84 83.1 77.5 85 NA 85.7 86 68.5NA 87 NA 77.5 88 39.2 77.4 89 87.0 NA 90 NA 91.4 91 94.4 NA 93 82.0 NA94 98.1 74.8 95 NA 86.9 98 73.4 NA 100 91.5 NA 103 72.5 83.2 106 96.3 NA107 94.5 NA 108 94.6 NA 109 80.6 NA 110 59.3 NA 111 74.7 NA 112 32.9 NA113 37.5 NA 114 98.4 NA 115 97.8 NA 116 93.3 NA 117  5.0 NA 118 12.8 NA119 95.7 NA 120 96.1 NA 121 71.2 NA 122 97.5 NA 123 83.8 NA 124 98.0 NA125 95.1 NA 126 97.4 NA 127 96.7 NA 128 86.2 NA 129 96.8 NA 130 97.8 NA131 96.4 NA 132 95.5 NA 133 63.3 NA 134 96.6 NA 135 96.2 NA 136 95.2 NA137 98.1 NA 138 92.9 NA 139 52.4 NA 141 94.9 NA 142 92.5 NA

Packaging of Plasmid DNA

All equipment and disposable supplies are certified free of RNaseactivity by the manufacturer or rendered RNase free by use of theRNaseZap reagent (LifeTechnologies). Plasmid is encapsulated at acationic lipid amine to DNA phosphate (N:P) molar ratio of 4:1. Lipids(cationic lipid, DSPC, cholesterol and lipidated PEG) are dissolved inethanol. The molar ratios are 40:10:38:2, respectively. The mixture issonicated briefly, then gently agitated for 5 minutes and thenmaintained at 37° C. until use. Plasmid is exchanged into citrate bufferpH 6.0 by use of Amicon Ultra-15 centrifugal concentrators, and thefinal concentration is adjusted to 0.053 mg/ml and held at 37° C. untiluse. An equal volume of lipids in ethanol, plasmid in citrate buffer,and citrate buffer alone are drawn into disposable syringes. Tubingleading from syringes containing lipids and DNA are attached to the Tjunction, and tubing leading from the syringe containing citrate bufferalone is paired with the tubing exiting the T-junction over a collectionvessel containing a stir bar on an active stir plate. Syringes areplaced in a syringe pump set to expel contents at a flow rate of 1 mlper minute. The pump is activated, and the collected DNA in lipidnanoparticles is transferred to SnakeSkin dialysis tubing (10,000 MWCO,Thermo Scientific). Material is dialyzed against RNAse- and pyrogen-free1× phosphate buffered saline overnight at 4° C.

Measurement of Plasmid DNA encapsulation

Percent encapsulation of plasmid DNA in lipid nanoparticles isdetermined using the Quant-iT Ribogreen RNA Assay kit (LifeTechnologies). The LNP-DNA suspension is assayed in buffer (DNA outsidethe particle), and buffer plus Triton X-100 detergent (total DNA). Thedifference calculated is the DNA inside the particle. Prepare a 1000ng/mL stock using unpackaged plasmid and use this to generate a standardcurve (0 ng/ml, 15.63-1000 ng/ml) in TE and TE+0.75% Triton X-100.Prepare LNP-plasmid samples in TE buffer and TE buffer+0.75% TritonX-100 with appropriate dilution so that reading is in the range ofstandard curve (25 fold). In a 384-well plate (Costar non-treated #3573)add 0.04 ml of standard (in duplicate) or sample (in triplicate) perwell. Dilute Ribogreen reagent 240-fold in TE buffer and add 0.06 ml perwell. Mix contents of wells and measure fluorescence (excitation=480 nm,emission=520 nm). Subtract background values (no DNA) from standard andtest sample values and determine the concentrations of DNA in thesamples using the standard curves. Determine the percent encapsulationof the sample by dividing the difference in concentrations betweensample+triton and sample in buffer alone by the sample+tritonconcentration.

Polydispersity Index (PDI) Measurements

Unless indicated otherwise, all PDIs referred to herein are the PDI ofthe fully formed nanoparticle, as measured by dynamic light scatteringon a Malvern Zetasizer. The nanoparticle sample was diluted in phosphatebuffered saline (PBS) so that the count rate was approximately 200-400kcts. The data is presented in Table 3 as a weighted average of theintensity measure.

The Particle Size of the Lipid Nanoparticle

Unless indicated otherwise, all particle size measurements referred toin Table 3 are the Z-average particle size of the fully formednanoparticle, as measured by dynamic light scattering on a MalvernZetasizer. The nanoparticle sample was diluted in phosphate bufferedsaline (PBS) so that the count rate is approximately 200-400 kcts.

Lipid Nanoparticle Characterization Data

TABLE 3 in vitro data on selected examples Example 1 Example 94 Example16 Example 80 Nucleic % Size % Size % Size % Size acid Encap. (nM) PDIEncap. (nM) PDI Encap. (nM) PDI Encap. (nM) PDI hLeptin 97.1 128.1 0.07198.1 119.1 0.101 98.1 122.6 0.103 mRNA hFIX 94.0 95.5 0.082 96.9 148.30.079 96.9 102.6 0.141 mRNA FLuc 94.7 108.8 0.100 93.7 129.2 0.093 96.8106.7 0.145 85.3 193.5 0.085 mRNA Gluc 97.3 84.7 0.093 95.9 76.9 0.11397.9 106.7 0.145 mRNA mEPO 95.8 104.1 0.073 96.4 108.5 0.099 97.9 83.20.112 96.2 166.0 0.083 mRNA hLep 93.5 109.8 0.138 95.0 109.8 0.153 96.7102.6 0.155 plasmid pDNA 94.3 110 0.135 89.3 123.5 0.152 96.4 101.60.128 FVII 74.8 122.6 0.073 88.9 95.7 0.075 85.2 97.3 0.051 siRNA

In Vivo Data Mouse Factor VII Dosing

Female CD-1 mice were received from Harlan Labs and maintained onstandard lab chow and water ad libitum. The animals weighedapproximately 25 gr at time of dosing. Formulated Factor VII siRNA wasadministered as a single dose intravenously via the lateral tail vein.Approximately 48 hours after injection, the mice were euthanized by CO₂inhalation followed by exsanguination through the vena cava. The bloodwas collected in tubes containing 0.105M sodium citrate anticoagulantfor plasma Factor VII activity analysis.

Factor VII Activity Assay

Plasma collected from injected mice was assayed for Factor VII enzymeactivity using the Biophen FVII kit from Hyphen Biomedical (catalognumber 221304). An assay standard curve was prepared using pooled plasmaaliquots from the vehicle control animals. All samples were diluted tofall within the linear range of the standard curve and Factor VIIactivity relative to control plasma was reported.

Lipid nanoparticles comprising lipid compounds of formula (I) and theFVII siRNA duplex sequence listed above were tested in the Factor VIIActivity Assay. The results of this assay are given in Table 3 4 belowas a percent knock down of plasma Factor VII enzyme activity at a doseof 0.3 mg/kg and 0.03 mg/kg.

Mouse EPO ELISA

A rat anti-mouse Erythropoietin antibody is coated on 384-well whitemicrotiter plates overnight, then blocked for assay. Then, plasmasamples are diluted in a casein-based sample diluent and incubated onthe plate with buffer controls and mouse EPO standards. The plate isthen washed to remove unbound material. A biotinylated rat anti-mouseErythropoietin antibody is then added to the plate to detect mouse EPObound by the capture antibody. The plate is washed again and astreptavidin-conjugated horseradish peroxidase reagent is incubated onthe plate. A third wash step is performed and a chemiluminescent reagentis added to the plate and immediately read by a capable plate readerusing all wavelengths and a 50 millisecond integration time. Unknownsamples are interpolated off the mouse EPO standard curve.

Human Factor IX ELISA

An sheep anti-human Factor IX antibody (Cat# FIX-EIA-C) from EnzymeResearch Laboratories is coated on 384-well white microtiter platesovernight at a concentration of 5 ug/ml, then blocked for assay usingKPL blocker (Cat#50-82-00). Then, plasma samples are diluted in acasein-based sample diluent and incubated on the plate with biologicalcontrols and a standard curve created from recombinant protein(Cat#HCIX-0040). The plate is then washed to remove unbound material. Abiotinylated sheep anti-human Factor IX antibody (Cat# FIX-EIA-D) fromEnzyme Research Laboratories is then added to the plate at 0.6 ug/ml todetect human Factor IX protein bound by the capture antibody. The plateis washed again and a streptavidin-conjugated horseradish peroxidasereagent (Cat#21140) diluted 1:1250, is incubated on the plate. A thirdwash step is performed and chemiluminescent reagents (Cat#1859678 &Cat#18596789) are combined and added to the plate and immediately readby a capable plate reader using all wavelengths and a 50 millisecondintegration time. Unknown samples are interpolated off the humanrecombinant Factor IX standard curve.

Casein Sample Diluent, pH 7.20

The Sample Diluent contains 0.7% Casein, 1.7 mM Sodium PhosphateMonobasic, 8.1 mM Sodium Phosphate Dibasic Heptahydrate, 0.15M SodiumChloride, 0.7% Triton X-100, and 0.1% Sodium Azide

Biotinylated Antibody Casein Diluent, pH 7.15

The diluent contains 0.4% Casein, 1.7 mM Sodium Phosphate Monobasic, 8.1mM Sodium Phosphate Dibasic Heptahydrate, 0.15M Sodium Chloride, and0.1% Sodium Azide

HRP Casein Diluent, pH 7.15

The diluent contains 0.4% Casein, 1.7 mM Sodium Phosphate Monobasic, 8.1mM Sodium Phosphate Dibasic Heptahydrate, 0.15M Sodium Chloride, and0.1% Chloroacetamide.Leptin

hLEPTIN

Human leptin in mouse plasma was measured by ELISA. Antibodies purchasedfrom the R&D Systems duoset (Cat # DY398E, part #840279 for captureantibody and part #840280 for detection antibody) were reconstitutedusing PBS and titered, again using PBS. The capture antibody was coatedat 4 ug/ml in 30 ul/well on a white Nunc® Maxisorp 384 well plate(Cat#460372). After an overnight incubation at room temperature thecapture antibody was aspirated and the plate blocked for 2 hours at roomtemperature with 90 ul/well of KPL milk blocker (Cat#50-82-00). Once theincubation was completed the plate was aspirated and recombinantstandards and samples were added to the plate at 30 ul/well for 2 hoursat 37° C. while shaking at 600 rpm. Sample/standard dilutions were madeusing casein sample diluent. Washing/aspiration 3 times with 100 ul/wellfollowed, using Teknova plate wash solution (Cat# P1192). Next,detection antibody was diluted using casein detection antibody diluentto 12.5 ng/ml and added at 30 ul/well for 2 hours room temperature.After this incubation, the plate was washed again and a solution ofpoly-streptavidin-HRP (Cat#21140) at a 1:1250 dilution in HRP dilutionbuffer was added to each well (30 ul/well) and incubated for 30 minutesroom temperature. A final wash/aspiration removed the HRP solution and achemiluminescent substrate was added at 30 ul/well (Cat#1859678 &1859679). The plate was quickly read using a SpectramaxM5 plate readerwith a 50 ms integration time. The dynamic range of the ELISA is from100-2,000 μg/ml (6.25-125 pM) of human leptin. The assay is applicableto plasma from mice, rats and cynomolgus monkeys.

Mouse Intravenous Tail Vein Injection of Modified Synthetic Leptin mRNA

Before the tail vein injection, mouse body weights were recorded anddiet weighted, with mice grouped according to their body weights. Micewere prepared by warming them under a heating lamp for ˜2 minutes, withthe mice about 12 inches from heat lamp.

For the tail vein injection procedure, the mice were placed in arestrainer and their tails cleaned with 70% alcohol. A 27 gauge needle(Becton Dickinson, Catalogue #305109) connected with a 1 ml syringe(Becton Dickinson, Catalogue #309659) was inserted into the tail vein,with bevel facing up, and the syringe plunger was pulled backwards toensure blood is drawn into the syringe. The desired volume of modifiedsynthetic leptin mRNA was injected by hand with moderate pressure andspeed. The needle was then withdrawn and bleeding stopped by addingpressure to injection site with gauze.

Single housed, 8-9 week old, male C57BL/6 mice were used for the in vivostudy. FPLC purified modified synthetic leptin mRNA (SEQ ID NO:6) inwhich the uridines were substituted with pseudouridine was packaged in acationic lipid (N:P molar ratio=8:1) and then were diluted in injectablesaline at a dose of 10 μg per average group body weight.

On day 0, animals were weighed and sorted according to average bodyweight. Mice were dosed, and food intake (FI) was recorded, on each ofdays 1-7 and days 9, 11, and 16.

Mouse Subcutaneous Injection of Modified Synthetic Leptin mRNA

Prior to subcutaneous injection, mouse body weights were recorded anddiet weighted, with mice grouped according to their body weights. Themice were manually restrained and placed on a work surface. Theirscruffs were pinched and lifted away from the underlying muscle, thespace into which was inserted a 25 gauge needle connected with a 1 mlsyringe. The syringe plunger was pulled backwards in such a way as toensure no fluid was drawn into the syringe, and then the desired volumeof leptin mRNA was hand injected with moderate pressure and speed. Theneedle was then withdrawn and the mice returned to their cages.

8-9 week old, male C57BL/6 mice were used for the in vivo study. FPLCpurified modified synthetic leptin mRNA (SEQ ID NO: 6) in which theuridines were substituted with pseudouridine (N:P molar ratio=8:1)packaged in multiple cationic lipid were diluted in injectable saline ata dose of 10 μg per average group body weight.

On day 0, animals were weighed and sorted according to average bodyweight. ice were dosed at 9 AM and blood was taken at 9 AM on day 0.Blood was also taken at 9 AM on each of days 1 and 2 and assessed forleptin protein levels. Body weight and food intake were also recorded.

TABLE 4 In vivo data Nucleic acid Example 1 Example 91 Example 16Example 80 Expression of protein (ng/ml) following IV injection ofencapsulated mRNA in C57B6 mice hLeptin 66.3 ng/mL 33.6 ng/mL 21.4 ng/mL(0.4mpk, C57) (0.2 mpk, ob/ob) (0.2 mpk, ob/ob) mEPO 247.6 ng/mL (0.2mpk, ob/ob) Expression of protein (ng/ml) following SC injection ofencapsulated mRNA in C57B6 mice hLeptin 16.2 ng/mL 3.5 ng/mL 8.4 ng/mL(0.2mpk, ob/ob) (0.2 mpk, ob/ob) (0.2 mpk, ob/ob) 1.2 ng/mL (0.2 mpk,C57) FVII knock-down (%) following IV injection of encapsulated siRNA inXXX mice FVII 97%/0.03 95%/0.03 mpk mpk

Immunogenicity Studies

Plasmid DNA encoding alphavirus replicons encoding the F protein of RSVas a transgene served as a template for synthesis of RNA in vitro. Thereplicons contain the alphavirus genetic elements required for RNAreplication but lack those encoding gene products necessary for particleassembly; the structural proteins are instead replaced by a protein ofinterest (e.g. an immunogen, such as full-length RSV F protein) and sothe replicons are incapable of inducing the generation of infectiousparticles. A T7 bacteriophage promoter upstream of the alphavirus cDNAfacilitates the synthesis of the replicon RNA in vitro. Other promoters,such as SP6 could be used as alternatives.

Transcriptions were performed for 2 hours at 37° C. in the presence of7.5 mM (T7 RNA polymerase) or 5 mM (SP6 RNA polymerase) of each of thenucleoside triphosphates (ATP, CTP, GTP and UTP) following theinstructions provided by the manufacturer (Ambion). Followingtranscription the template DNA was digested with TURBO DNase (Ambion).The replicon RNA was precipitated with LiCl and reconstituted innuclease-free water. Uncapped RNA was capped post-transcriptionally withVaccinia Capping Enzyme (VCE) using the ScriptCap m7G Capping System(Epicentre Biotechnologies) as outlined in the user manual; repliconscapped in this way are given the “v” prefix e.g. vA317 is the A317replicon capped by VCE. Post-transcriptionally capped RNA wasprecipitated with LiCl and reconstituted in nuclease-free water. Theconcentration of the RNA samples was determined by measuringOD_(260 nm). Integrity of the in vitro transcripts was confirmed bydenaturing agarose gel electrophoresis.

Encapsulation in DlinDMA-Based Liposomes

RNA was encapsulated in liposomes made essentially by the method ofGeall et al. (2012) PNAS vol. 109 (36): 14604-14609, Jeffs et al. (2005)Pharmaceutical Research 22 (3):362-372 and Maurer et al. (2001)Biophysical Journal, 80: 2310-2326 The liposomes were made of 10% DSPC(zwitterionic), 40% cationic lipid, 48% cholesterol and 2%PEG-conjugated DMG (2 kDa PEG). These proportions refer to the % molesin the total liposome.

DSPC (1,2-Diastearoyl-sn-glycero-3-phosphocholine) was purchased fromGenzyme. Cholesterol was obtained from Sigma-Aldrich. PEG-conjugated DMG(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol), ammonium salt), DOTAP(1,2-dioleoyl-3-trimethylammonium-propane, chloride salt) and DC-chol(3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride)were from Avanti Polar Lipids.

Briefly, lipids were dissolved in ethanol (2 ml), a RNA replicon wasdissolved in buffer (2 ml, 100 mM sodium citrate, pH 6) and these weremixed with 2 ml of buffer followed by 1 hour of equilibration. Themixture was then dialized overnight against 1×PBS. The resulting productcontained liposomes, with ˜70-95% encapsulation efficiency. For in vitroand in vivo experiments formulations were diluted to the required RNAconcentration with 1×PBS.

The percentage of encapsulated RNA and RNA concentration were determinedby Quant-iT RiboGreen RNA reagent kit (Invitrogen), followingmanufacturer's instructions. The ribosomal RNA standard provided in thekit was used to generate a standard curve. Liposomes were diluted 10× or100× in 1×TE buffer (from kit) before addition of the dye. Separately,liposomes were diluted 10× or 100× in 1×TE buffer containing 0.5% TritonX before addition of the dye (to disrupt the liposomes and thus to assaytotal RNA). Thereafter an equal amount of dye was added to each solutionand then ˜180 μL of each solution after dye addition was loaded induplicate into a 96 well tissue culture plate. The fluorescence (Ex 485nm, Em 528 nm) was read on a microplate reader. All liposomeformulations were dosed in vivo based on the encapsulated amount of RNA.

RSV Immunogenicity

A self-replicating replicon encoding RSV F protein was administered toBALB/c mice, 8 animals per group, by bilateral intramuscularvaccinations (50 μL per leg) on days 0 and 21 with the replicon (0.1 and1 ng) formulated as liposomes with the lipids described below. Allliposomes tested were composed of 40% cationic lipid, 10% DSPC, 48%cholesterol and 2% PEG-DMG with similar amounts of RNA. The liposomeswere all prepared using the same technique as described above.

% entrapment (% E), Concentration of RNA (Conc), particle size measuredby DLS, and polydispersity reported by DLS of LNPs prepared withdifferent cationic lipids

Conc % E (ug/mL) Size (nm) PDI ex- 1st 2nd 1st 2nd 1st 2nd 1st 2nd ampleIM IM IM IM IM IM IM IM 1 91.11 87.13 11.824 9.475 139.1 131.3 0.1050.045 18 96.15 96.25 10.225 11.484 132.2 139.8 0.13 0.065 105 85.2269.54 9.804 9.929 137.2 135.7 0.107 0.123 98 85.91 76.36 10.388 10.128139.4 130.9 0.07 0.065 58 87.48 87.03 12.16 12.309 139.4 137.1 0.1110.075 70 90.75 94.24 12.099 10.324 133.7 141.7 0.121 0.118 115 95.7695.78 9.013 9.336 141.3 130.9 0.075 0.065 131 85.31 80.31 10.153 9.325138.3 143.5 0.119 0.07Immunogenicity data two weeks after two immunizations of vA375 RNAexpressing RSV-F of LNPs prepared with different cationic lipids. LNPswere prepared fresh for each immunization.

Immunogenicity (2wp2, log10 IgG titers) Lipid ID 1 ng 0.1 ng 1 4.22 3.9818 4.26 3.5 105 4.53 4.21 98 4.64 4.28 58 4.26 4.19 70 4.37 3.90 1153.31 1.91 131 5.22 4.43

It should be understood that for all numerical bounds describing someparameter in this application, such as “about,” “at least,” “less than,”and “more than,” the description also necessarily encompasses any rangebounded by the recited values. Accordingly, for example, the description“at least 1, 2, 3, 4, or 5” also describes, inter alia, the ranges 1-2,1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, et cetera.

For all patents, applications, or other reference cited herein, such asnon-patent literature and reference sequence information, it should beunderstood that they are incorporated by reference in their entirety forall purposes as well as for the proposition that is recited. Where anyconflict exists between a document incorporated by reference and thepresent application, this application will control. All informationassociated with reference gene sequences disclosed in this application,such as GeneIDs or accession numbers (typically referencing NCBIaccession numbers), including, for example, genomic loci, genomicsequences, functional annotations, allelic variants, and reference mRNA(including, e.g., exon boundaries or response elements) and proteinsequences (such as conserved domain structures), as well as chemicalreferences (e.g., PubChem compound, PubChem substance, or PubChemBioassay entries, including the annotations therein, such as structuresand assays, et cetera), are hereby incorporated by reference in theirentirety.

Headings used in this application are for convenience only and do notaffect the interpretation of this application.

Preferred features of each of the aspects provided by the invention areapplicable to all of the other aspects of the invention mutatis mutandisand, without limitation, are exemplified by the dependent claims andalso encompass combinations and permutations of individual features(e.g., elements, including numerical ranges and exemplary embodiments)of particular embodiments and aspects of the invention, including theworking examples. For example, particular experimental parametersexemplified in the working examples can be adapted for use in theclaimed invention piecemeal without departing from the invention. Forexample, for materials that are disclosed, while specific reference ofeach of the various individual and collective combinations andpermutations of these compounds may not be explicitly disclosed, each isspecifically contemplated and described herein. Thus, if a class ofelements A, B, and C are disclosed as well as a class of elements D, E,and F and an example of a combination of elements A-D is disclosed,then, even if each is not individually recited, each is individually andcollectively contemplated. Thus, in this example, each of thecombinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C—F are specificallycontemplated and should be considered disclosed from disclosure of A, B,and C; D, E, and F; and the example combination A-D. Likewise, anysubset or combination of these is also specifically contemplated anddisclosed. Thus, for example, the sub-groups of A-E, B-F, and C-E arespecifically contemplated and should be considered disclosed fromdisclosure of A, B, and C; D, E, and F; and the example combination A-D.This concept applies to all aspects of this application, includingelements of a composition of matter and steps of method of making orusing the compositions.

The forgoing aspects of the invention, as recognized by the personhaving ordinary skill in the art following the teachings of thespecification, can be claimed in any combination or permutation to theextent that they are novel and non-obvious over the prior art-thus, tothe extent an element is described in one or more references known tothe person having ordinary skill in the art, they may be excluded fromthe claimed invention by, inter alia, a negative proviso or disclaimerof the feature or combination of features.

1. A compound, or salt thereof, of formula (I):

n is 0, 1, 2, 3 or 4; p is 0, 1, 2, 3, 4, 5, 6, 7 or 8; L₁ is —O— or abond; L₂ is —OC(O)— or —C(O)O—; R¹ is selected from:

v is 0, 1, 2, 3 or 4; w is 0, 1, 2, or 3; Cyl is 5-7 membered nitrogencontaining heterocycle optionally substituted with one or two alkylgroups; Ar is an aryl group, optionally substituted with C₁₋₈ alkylamino group; R and R′ are each, independently, hydrogen or C₁₋₈ alkyl;and R² is selected from C₆₋₂₀ alkyl optionally substituted with ahydroxyl, C₁₅₋₁₉ alkenyl, C₁₋₁₂alkyl-OC(O)—C₅₋₂₀alkyl,C₁₋₁₂alkyl-C(O)O—C₅₋₂₀alkyl and

R³ is selected from: C₄₋₂₂ alkyl, C₁₂₋₂₂ alkenyl,


2. The compound, or salt thereof, according to claim 1, wherein thecompound is of formula (II):


3. The compound, or salt thereof, according to claim 1, wherein thecompound is of formula (III):


4. The compound, or salt thereof, according to claim 1, wherein R² isselected from:


5. The compound, or salt thereof, of claim 1, wherein the compound is offormula (IV):


6. The compound, or salt thereof, according to claim 1, wherein R³ isselected from:


7. The compound, or salt thereof, according to claim 1, wherein thecompound is of formula (V):


8. The compound, or salt thereof, according to claim 1, wherein thecompound is of formula (VI):


9. The compound, or salt thereof, according to claim 1, wherein thecompound is of formula (VII):


10. The compound, or salt thereof, of claim 1, wherein R¹ is selectedfrom:


11. The compound, or salt thereof, of claim 1, wherein R¹ is


12. The compound, or salt thereof, according to claim 1, wherein thecompound is selected from:2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azaoctadecan-18-yl)propane-1,3-diyldioctanoate;2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2-azaheptadecan-17-yl)propane-1,3-diyldioctanoate;2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2-azapentadecan-15-yl)propane-1,3-diyldioctanoate;2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azahexadecan-16-yl)propane-1,3-diyldioctanoate;2-(8-dodecyl-2-methyl-6,12-dioxo-5,7,11-trioxa-2-azaheptadecan-17-yl)propane-1,3-diyldioctanoate;2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azanonadecan-19-yl)propane-1,3-diyldioctanoate;2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2-azaoctadecan-18-yl)propane-1,3-diyldioctanoate;2-(8-dodecyl-2-methyl-6,12-dioxo-5,7,11-trioxa-2-azaoctadecan-18-yl)propane-1,3-diyldioctanoate;2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azaicosan-20-yl)propane-1,3-diyldioctanoate;2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2-azanonadecan-19-yl)propane-1,3-diyldioctanoate; 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl4,4-bis(octyloxy)butanoate;3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl4,4-bis((2-ethylhexyl)oxy)butanoate;3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl4,4-bis((2-propylpentyl)oxy)butanoate;3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl4,4-bis((2-propylpentyl)oxy)butanoate;3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl4,4-bis((2-propylpentyl)oxy)butanoate;3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate;3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl6,6-bis(hexyloxy)hexanoate;3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl6,6-bis((2-ethylhexyl)oxy)hexanoate;3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl8,8-bis(hexyloxy)octanoate;3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl8,8-dibutoxyoctanoate;3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl8,8-bis((2-propylpentyl)oxy)octanoate;3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl8,8-bis((2-propylpentyl)oxy)octanoate;3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl8,8-bis((2-propylpentyl)oxy)octanoate;3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl3-octylundecanoate;3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl3-octylundec-2-enoate;3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl7-hexyltridec-6-enoate;3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl9-pentyltetradecanoate;3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl9-pentyltetradec-8-enoate;3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl5-heptyldodecanoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)tridecyl5-heptyldodecanoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)undecyl5-heptyldodecanoate; 1,3-bis(octanoyloxy)propan-2-yl(3-(((2-(dimethylamino)ethoxy)carbonyl)oxy)pentadecyl) succinate;1,3-bis(octanoyloxy)propan-2-yl(3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl) succinate;1-(3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl) 10-octyldecanedioate; 1-(3-(((3-(diethylamino) propoxy)carbonyl)oxy)pentadecyl)10-octyl decanedioate;1-(3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl) 10-octyldecanedioate; 1-(3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl)10-(2-ethylhexyl) decanedioate;1-(3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl)10-(2-ethylhexyl) decanedioate;3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl10-(octanoyloxy)decanoate;8-dodecyl-2-methyl-6,12-dioxo-5,7,11-trioxa-2-azanonadecan-19-yldecanoate; 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl10-(octanoyloxy)decanoate;3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl10-(octanoyloxy)decanoate; (9Z,12Z)-3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl octadeca-9,12-dienoate;(9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyloctadeca-9,12-dienoate;(9Z,12Z)-3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyloctadeca-9,12-dienoate;(9Z,12Z)-3-(((2-(dimethylamino)ethoxy)carbonyl)oxy)pentadecyloctadeca-9,12-dienoate; 1-((9Z,12Z)-octadeca-9,12-dienoyloxy)pentadecan-3-yl 1,4-dimethylpiperidine-4-carboxylate;2-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecyl4,4-bis((2-ethylhexyl)oxy)butanoate;(9Z,12Z)-(12Z,15Z)-3-((3-(dimethylamino)propanoyl)oxy)henicosa-12,15-dien-1-yloctadeca-9,12-dienoate;(12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)henicosa-12,15-dien-1-yl3-octylundecanoate;(12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)henicosa-12,15-dien-1-yl5-heptyldodecanoate;(12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)henicosa-12,15-dien-1-yl7-hexyltridecanoate;(12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)henicosa-12,15-dien-1-yl9-pentyltetradecanoate;(12Z,15Z)-1-((((9Z,12Z)-octadeca-9,12-dien-1-yloxy)carbonyl)oxy)henicosa-12,15-dien-3-yl 3-(dimethylamino)propanoate;(13Z,16Z)-4-(((2-(dimethylamino)ethoxy)carbonyl)oxy)docosa-13,16-dien-1-yl2,2-bis(heptyloxy)acetate;(13Z,16Z)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)docosa-13,16-dien-1-yl2,2-bis(heptyloxy)acetate; 2,2-bis(heptyloxy)ethyl3-((3-ethyl-10-((9Z,12Z)-octadeca-9,12-dien-1-yl)-8,15-dioxo-7,9,14-trioxa-3-azaheptadecan-17-yl)disulfanyl)propanoate;(13Z,16Z)-4-(((3-(dimethylamino)propoxy)carbonyl)oxy)docosa-13,16-dien-1-ylheptadecan-9-yl succinate;(9Z,12Z)-2-(((11Z,14Z)-2-((3-(dimethylamino)propanoyl)oxy)icosa-11,14-dien-1-yl)oxy)ethyloctadeca-9,12-dienoate;(9Z,12Z)-3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyloctadeca-9,12-dienoate;3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl3-octylundecanoate;3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-hydroxytridecyl5-heptyldodecanoate;3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl5-heptyldodecanoate;3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl7-hexyltridecanoate;3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-hydroxytridecyl9-pentyltetradecanoate;3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl9-pentyltetradecanoate;1-(3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl)10-octyl decanedioate;3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl10-(octanoyloxy)decanoate; (9Z,12Z)-3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-5-octyltridecyloctadeca-9,12-dienoate;3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-5-octyltridecyl decanoate;5-(((3-(dimethylamino)propoxy)carbonyl)oxy)-7-octylpentadecyl octanoate;(9Z,12Z)-5-(((3-(dimethylamino) propoxy)carbonyl)oxy)-7-octylpentadecyloctadeca-9,12-dienoate;9-(((3-(dimethylamino)propoxy)carbonyl)oxy)-11-octylnonadecyl octanoate;9-(((3-(dimethylamino)propoxy)carbonyl)oxy)-11-octylnonadecyl decanoate;(9Z,12Z)-9-(((3-(di methylamino)propoxy)carbonyl)oxy)nonadecyloctadeca-9,12-dienoate;9-(((3-(dimethylamino)propoxy)carbonyl)oxy)nonadecyl hexanoate;9-(((3-(dimethylamino)propoxy)carbonyl)oxy)nonadecyl 3-octylundecanoate;9-((4-(dimethylamino)butanoyl)oxy) nonadecyl hexanoate;9-((4-(dimethylamino)butanoyl)oxy) nonadecyl 3-octylundecanoate;(9Z,9′Z,12Z,12′Z)-2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diylbis(octadeca-9,12-dienoate);(9Z,9′Z,12Z,12′Z)-2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl bis(octadeca-9,12-dienoate);(9Z,9′Z,12Z,12′Z,15Z,15′Z)-2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl bis(octadeca-9,12,15-trienoate);(Z)-2-((4-(((3-(dimethylamino) propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl dioleate; 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy) hexadecanoyl)oxy)propane-1,3-diylditetradecanoate;2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diylditetradecanoate;2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl ditetradecanoate;2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyldidodecanoate; 2-((4-(((3-(diethylamino) propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl didodecanoate;2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl didodecanoate; 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy) hexadecanoyl)oxy)propane-1,3-diyl bis(decanoate);2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl bis(decanoate); 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy) hexadecanoyl)oxy)propane-1,3-diyl dioctanoate;2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl dioctanoate;2-(((13Z,16Z)-4-(((3-(dimethylamino)propoxy)carbonyl)oxy)docosa-13,16-dienoyl)oxy)propane-1,3-diyldioctanoate;2-(((13Z,16Z)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)docosa-13,16-dienoyl)oxy)propane-1,3-diyldioctanoate;(9Z,9′Z,12Z,12′Z)-2-((2-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecanoyl)oxy)propane-1,3-diyl bis(octadeca-9,12-dienoate);(9Z,9′Z,12Z,12′Z)-2-((2-(((3-(dimethylamino)propoxy)carbonyl)oxy)dodecanoyl)oxy)propane-1,3-diylbis(octadeca-9,12-dienoate);(9Z,9′Z,12Z,12′Z)-2-((2-(((3-(dimethylamino)propoxy)carbonyl)oxy)tetradecanoyl)oxy)propane-1,3-diylbis(octadeca-9,12-dienoate);(9Z,9′Z,12Z,12′Z)-2-((2-(((3-(diethylamino)propoxy)carbonyl)oxy)dodecanoyl)oxy)propane-1,3-diylbis(octadeca-9,12-dienoate);2-((2-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecanoyl)oxy)propane-1,3-diyldioctanoate; 4,4-bis(octyloxy)butyl4-(((3-(dimethylamino)propoxy)carbonyl)oxy) hexadecanoate;4,4-bis(octyloxy)butyl 2-(((3-(diethylamino)propoxy)carbonyl)oxy)dodecanoate;(9Z,12Z)-10-dodecyl-3-ethyl-14-(2-((9Z,12Z)-octadeca-9,12-dienoyloxy)ethyl)-8,13-dioxo-7,9-dioxa-3,14-diazahexadecan-16-yloctadeca-9,12-dienoate; 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)-11-(octanoyloxy)undecanoyl)oxy)propane-1,3-diyldioctanoate;(9Z,9′Z,12Z,12′Z)-2-(9-dodecyl-2-methyl-7,12-dioxo-6,8,13-trioxa-2-azatetradecan-14-yl)propane-1,3-diylbis(octadeca-9,12-dienoate);3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl4,4-bis(octyloxy)butanoate;3-(((3-(piperidin-1-yl)propoxy)carbonyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate;3-(((3-(piperazin-1-yl)propoxy)carbonyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate; 3-(((4-(diethylamino)butoxy)carbonyl)oxy)pentadecyl 6,6-bis(octyloxy)hexanoate;3-(((3-(4-methylpiperazin-1-yl)propoxy)carbonyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate;3-((((1-methylpiperidin-4-yl)methoxy)carbonyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate; 3-(((3-morpholinopropoxy)carbonyl)oxy)pentadecyl 6,6-bis(octyloxy) hexanoate;3-(((2-(diethylamino)ethoxy)carbonyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate;3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate;3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl6,6-bis((2-propylpentyl)oxy)hexanoate;3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl6,6-bis((2-propylpentyl)oxy)hexanoate LXR420:3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl6,6-bis((3-ethylpentyl)oxy)hexanoate;(2R)-1-((6,6-bis(octyloxy)hexanoyl)oxy) pentadecan-3-yl 1-methylpyrrolidine-2-carboxylate;(2S)-1-((6,6-bis(octyloxy)hexanoyl)oxy)pentadecan-3-yl1-methylpyrrolidine-2-carboxylate; (2R)-1-((6,6-bis(octyloxy)hexanoyl)oxy)pentadecan-3-yl pyrrolidine-2-carboxylate;1-((6,6-bis(octyloxy)hexanoyl)oxy) pentadecan-3-yl 1,3-dimethylpyrrolidine-3-carboxylate;3-((3-(1-methylpiperidin-4-yl)propanoyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate; 1-((6,6-bis(octyloxy)hexanoyl)oxy)pentadecan-3-yl 1,4-dimethylpiperidine-4-carboxylate;3-((5-(diethylamino)pentanoyl)oxy) pentadecyl 6,6-bis(octyloxy)hexanoate; 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl5-(4,6-diheptyl-1,3-dioxan-2-yl)pentanoate;3-(((3-(diethylamino)propoxy)carbonyl)oxy)undecyl6,6-bis(octyloxy)hexanoate;3-(((3-(diethylamino)propoxy)carbonyl)oxy)tridecyl6,6-bis(octyloxy)hexanoate;(12Z,15Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)henicosa-12,15-dien-1-yl 6,6-bis(octyloxy)hexanoate;6-((6,6-bis(octyloxy)hexanoyl)oxy)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexyl octanoate; 4,4-bis(octyloxy)butyl5-(((3-(diethylamino)propoxy)carbonyl)oxy)heptadecanoate;4,4-bis(octyloxy)butyl (3-(diethylamino)propyl) pentadecane-1,3-diyldicarbonate; 2-(5-((4-((1,4-dimethylpiperidine-4-carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyl dioctanoate;2-(5-((4-((1,3-dimethylpyrrolidine-3-carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyldioctanoate;2-(5-oxo-5-((4-(((S)-pyrrolidine-2-carbonyl)oxy)hexadecyl)oxy)pentyl)propane-1,3-diyl dioctanoate; 2-(5-((4-(((((S)-1-methylpyrrolidin-3-yl)oxy)carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyldioctanoate;2-(5-((4-(((((R)-1-methylpyrrolidin-3-yl)oxy)carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyldioctanoate; 2-(5-((4-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyl dioctanoate;2-(5-((4-((((1-methylpiperidin-4-yl)oxy)carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyldioctanoate;2-(10-dodecyl-3-ethyl-8,15-dioxo-7,9,14-trioxa-3-azanonadecan-19-yl)propane-1,3-diyldioctanoate;2-(11-dodecyl-3-ethyl-9,15-dioxo-8,10,14-trioxa-3-azanonadecan-19-yl)propane-1,3-diyldioctanoate;2-(5-((3-(((3-(1H-imidazol-1-yl)propoxy)carbonyl)oxy)pentadecyl)oxy)-5-oxopentyl)propane-1,3-diyldioctanoate;2-(5-oxo-5-((3-(((3-(piperidin-1-yl)propoxy)carbonyl)oxy)pentadecyl)oxy)pentyl)propane-1,3-diyldioctanoate; and2-(12-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azaoctadecan-18-yl)propane-1,3-diyldioctanoate.
 13. A lipid composition comprising a compound according toclaim 1, or a pharmaceutically acceptable salt thereof.
 14. The lipidcomposition according to claim 13, further comprising a biologicallyactive agent.
 15. The lipid composition according to claim 13, whereinthe lipid composition is in the form of a lipid nanoparticle.
 16. Apharmaceutical composition comprising a lipid composition according toclaim 13, and a pharmaceutically acceptable carrier or excipient.
 17. Amethod for the treatment of a disease or condition comprising the stepof administering a therapeutically effective amount of lipid compositionaccording to claim 14, to a patient in need of treatment thereof. 18.The composition of claim 14, wherein the composition comprises a RNAmolecule that encodes an immunogen, optionally wherein the RNA is a selfreplicating RNA.
 19. The composition of claim 18, wherein the immunogencan elicit an immune response in vivo against a bacterium, a virus, afungus or a parasite.
 20. A method for inducing an immune response to animmunogen in a vertebrate, comprising administering an effective amountof the composition of claim 18 to the vertebrate.